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Mechanisms of Chromosomal DNA Replication

Part 1: Mechanisms of Chromosomal DNA Replication: The DNA Replication Fork

00:00:07;29 Hi. I'm Steve Bell.
00:00:09;15 I'm a professor of biology at MIT
00:00:12;22 and an investigator of the Howard Hughes Medical Institute.
00:00:15;06 And what I'd like to tell you about today
00:00:16;19 is some of what we know about
00:00:18;28 the mechanisms of chromosomal DNA replication.
00:00:21;11 Now, this event is primarily mediated
00:00:24;02 by the function of a complex
00:00:25;20 multi-enzyme machines called replisomes,
00:00:28;18 that include three DNA polymerases,
00:00:30;08 an RNA polymerase,
00:00:31;21 as well as a DNA helicase.
00:00:34;06 And together these enzymes
00:00:36;28 must act to accurately, completely,
00:00:39;12 and rapidly replicate the genomic DNA.
00:00:42;26 The rapidity can be
00:00:45;28 illustrated by the fact that it moves...
00:00:48;03 these replisomes can move at up to
00:00:49;27 1000 base pairs per second,
00:00:51;09 and also that entire genomes
00:00:53;20 can be duplicated in as little as three or four minutes.
00:00:58;20 The accuracy is about 1 mistake
00:01:01;03 in every 10^10 base pairs.
00:01:02;23 To put that into perspective,
00:01:04;02 that would mean that if you typed
00:01:05;29 60 words per minute
00:01:07;17 for 38 years,
00:01:09;07 continuously,
00:01:10;14 you would have a single typographical error in the document you made...
00:01:14;09 so that's pretty impressive.
00:01:15;27 And finally, it's really important
00:01:18;04 that genomic replication be complete,
00:01:20;06 because at the end of genomic replication
00:01:22;13 comes cell division,
00:01:23;22 and you need a full copy of the genes
00:01:26;15 for both of the daughter cells,
00:01:28;12 so, if you do incomplete replication,
00:01:30;00 someone is not going to get their full complement of genes.
00:01:33;23 It's also worth noting that
00:01:35;21 when you try and segregate the chromosomes
00:01:37;21 into two cells
00:01:39;12 when they're not completely replicated,
00:01:40;24 this will lead to double-stranded breaks,
00:01:42;14 which can be both mutagenic
00:01:44;05 and, in some cases, lethal.
00:01:46;28 Now, it's sometimes hard to appreciate
00:01:49;18 how much DNA replication goes on
00:01:52;08 inside our bodies,
00:01:53;11 but the next slide
00:01:56;08 is sort of an attempt to make that clear to you.
00:01:57;17 So, many of you may know that
00:02:00;26 in each one of your cells,
00:02:02;15 there's approximately 2 meters of DNA,
00:02:03;29 and some of you may know that there's...
00:02:06;25 in your entire body,
00:02:08;05 there's approximately 150 million kilometers,
00:02:11;12 and that's enough DNA to go from the sun to the Earth.
00:02:15;18 Now, sticking with the astronomical sense,
00:02:17;14 the most remarkable number is
00:02:20;00 not how much is in your body right now,
00:02:21;20 but the amount that you will synthesize in your lifetime,
00:02:24;18 which is upwards of a light year of DNA.
00:02:29;03 And, if you think about that,
00:02:30;05 if you're not sort of up on your astronomical units,
00:02:32;17 that's 9.5 trillion kilometers of DNA.
00:02:36;06 And so the mere fact that I can stand here
00:02:38;12 and tell you about DNA replication,
00:02:40;18 and not be just
00:02:43;12 a bumbling mass of mutagenized cells,
00:02:44;24 is a testament to both the accuracy
00:02:46;27 and efficiency of this process.
00:02:49;13 Okay, so I'm just going to show you an animation, here,
00:02:52;07 of the process of DNA replication
00:02:54;19 at the E. coli replication fork.
00:02:58;00 And what I want to do over the next few minutes
00:03:00;12 is to tell you about the various enzymes
00:03:02;21 that are working together in this animation
00:03:05;10 so that you understand
00:03:07;13 exactly what's happening during this process.
00:03:11;10 So, the first thing I want to do
00:03:14;18 is tell you a little bit of the ground rules.
00:03:16;00 So, DNA polymerases
00:03:17;27 always extend the 3' end
00:03:20;07 of a growing DNA chain,
00:03:23;05 and this can be either
00:03:25;15 extending the 3' end of a DNA chain
00:03:28;12 or, it turns out,
00:03:30;07 it can also extend the 3' end of an RNA chain.
00:03:33;25 And in each case,
00:03:35;28 it extends this by reading
00:03:38;03 an oppositely-oriented template strand,
00:03:40;05 and in fact the process...
00:03:42;01 or, the place at which DNA polymerases start DNA synthesis
00:03:45;12 is called a primer-template junction,
00:03:47;15 and I'll be using that term
00:03:49;16 throughout the next few minutes
00:03:51;19 as I describe the function of these enzymes.
00:03:53;18 Now, both strands of the DNA
00:03:56;01 are replicated simultaneously
00:03:58;06 during the replication of chromosomes,
00:04:01;20 and this is to reduce the amount
00:04:03;25 of single-stranded DNA,
00:04:05;14 which is much more prone to chromosome breakage
00:04:07;20 than is double-stranded DNA.
00:04:10;09 Now, there's two different types of DNA polymerases
00:04:13;12 acting at this time.
00:04:15;22 One is called a leading-strand DNA polymerase,
00:04:18;11 and it acts by extending
00:04:21;16 the leading strand DNA
00:04:23;15 towards the unreplicated DNA,
00:04:24;27 or in the same direction
00:04:27;03 as the overall DNA replication process.
00:04:30;15 So, this is very easy to understand.
00:04:32;14 It's going to follow right behind
00:04:36;04 the unreplicated and un...
00:04:37;26 and double-stranded form of the DNA.
00:04:41;01 In contrast,
00:04:43;02 replication of the opposite strand
00:04:45;01 has to move in the opposite direction,
00:04:46;29 away from the direction of overall fork movement
00:04:50;11 or replication.
00:04:52;01 And so, in this instance,
00:04:53;14 the primers will be formed
00:04:55;28 and the polymerase will move
00:04:58;06 away from the overall unreplicated DNA,
00:05:01;21 and in the opposite direction of the fork movement.
00:05:04;25 Now, these two events are happening simultaneously
00:05:07;17 at the replication fork,
00:05:09;27 as is illustrated here,
00:05:12;10 and you can see that the lagging strand DNA polymerase
00:05:14;06 is moving in one direction,
00:05:15;23 the leading strand is moving in the opposite direction.
00:05:18;22 And when you finish an Okazaki fragment,
00:05:21;06 as these smaller fragments that are made
00:05:24;08 on the lagging strand are called,
00:05:25;23 you then reposition the polymerase
00:05:28;23 and start a new Okazaki fragment,
00:05:30;24 and, eventually, at the end of the replication process,
00:05:35;05 these primers that are used,
00:05:37;16 which I'll have more to say about in a moment,
00:05:39;09 have to be removed,
00:05:40;22 and the DNA linked together
00:05:42;26 to form a continuous strand,
00:05:44;25 unlike the leading strand,
00:05:46;18 where it is continuously synthesized.
00:05:48;23 Now, one property of DNA polymerases,
00:05:51;23 all DNA polymerases,
00:05:53;06 is that they cannot start a new DNA strand
00:05:56;02 by joining two deoxynucleotide triphosphates.
00:06:00;28 They have to have a primer,
00:06:04;04 in the form of a primer-template junction.
00:06:06;06 And so, in order to initiate the new strands
00:06:09;09 that are required for the replication process,
00:06:10;26 we need a different enzyme
00:06:12;21 called DNA primase.
00:06:14;10 And what DNA primase does is
00:06:17;13 it synthesizes RNA primers.
00:06:20;05 And this is because, unlike DNA polymerases,
00:06:22;29 RNA polymerase,
00:06:24;28 of which DNA primase is a specialized form of,
00:06:27;15 can take two ribonucleotides
00:06:29;24 and initiate a new strand of DNA.
00:06:33;18 Okay.
00:06:34;19 Now, importantly, once it does this
00:06:36;13 it can be extended by the DNA polymerase
00:06:38;12 -- it's basically forming a primer-template junction --
00:06:40;29 and one interesting property of the DNA primase in E. coli
00:06:44;29 is that it is stimulated to act
00:06:48;13 by interacting with another important protein
00:06:50;19 that acts at the replication form,
00:06:53;02 called the DNA helicase.
00:06:55;24 Now, replicative DNA helicases
00:06:57;14 always come in the form of
00:06:59;17 hexameric, ring-shaped structures,
00:07:02;24 as you see, here, okay.
00:07:04;06 And these hexameric ring-shaped structures
00:07:06;16 will encircle one of the two strands of the DNA,
00:07:10;08 and they will then move
00:07:12;28 in an ATP binding
00:07:15;12 and ATP hydrolysis-dependent fashion,
00:07:17;00 in a defined direction
00:07:19;22 along this single-stranded DNA,
00:07:21;18 and by doing so they will
00:07:24;13 displace the other strand of DNA.
00:07:25;24 So, you can see that, here,
00:07:27;07 with the helicase unwinding the DNA.
00:07:29;20 Now, I've looped this,
00:07:31;07 so you'll see it a few times,
00:07:32;19 but I want to point out, also,
00:07:34;08 that the direction that a helicase moves
00:07:36;29 on its encircled strand
00:07:39;11 is a property of the helicase,
00:07:40;20 and in this particular case
00:07:42;05 I've illustrated the E. coli
00:07:45;08 replicative DNA helicase, called DnaB.
00:07:47;12 And its polarity, as this property is called,
00:07:50;12 is in the 5'-to-3' direction,
00:07:52;28 so you can see it's starting at the 5' end
00:07:57;08 and moving towards the 3' end of the DNA.
00:07:59;26 Now, we've talked about
00:08:01;27 a number of proteins that are involved at this point,
00:08:04;04 but one that is not an enzyme,
00:08:05;23 unlike the ones we've talked about thus far,
00:08:08;05 has a primary role of holding the two strands apart
00:08:11;20 after you unwind.
00:08:13;02 Because, of course, these two strands of DNA that I have over here
00:08:17;08 are in fact complementary to one another,
00:08:19;18 and could rapidly reanneal.
00:08:22;01 Now, this is prevented by two different events.
00:08:24;14 The first one is simple to understand
00:08:26;29 -- that is, that the leading strand DNA polymerase,
00:08:30;02 up here at the top,
00:08:31;28 follows almost directly behind the helicase.
00:08:34;17 And so, that single-stranded DNA
00:08:36;29 is very rapidly converted into double-stranded DNA,
00:08:40;09 and this prevents it from annealing
00:08:42;20 with the complementary lagging strand template.
00:08:44;24 Now, there's another concern, however,
00:08:47;12 which is that the lagging strand template
00:08:49;01 will anneal on itself,
00:08:50;22 and so there are a specific set of proteins
00:08:52;26 called single-stranded DNA binding proteins,
00:08:54;29 or SSBs,
00:08:56;14 that will bind the single-stranded region
00:08:59;11 of the lagging template,
00:09:01;03 and hold it in a single-stranded state,
00:09:02;28 preventing it from annealing to itself.
00:09:06;29 And what's important about this is
00:09:09;16 not only does it keep it from reannealing,
00:09:11;13 but when a DNA polymerase approaches
00:09:13;21 a region of single-stranded bound...
00:09:16;03 single-stranded DNA bound by the SSB,
00:09:19;11 it is readily displaced,
00:09:21;27 allowing the template that's left behind
00:09:24;04 to be readily replicated by the polymerase.
00:09:27;28 Okay.
00:09:29;20 So, we've talked about the leading and lagging polymerases,
00:09:33;04 but it turns out, at the replication fork,
00:09:35;05 they're part of a larger complex
00:09:37;18 called a holoenzyme, in particular,
00:09:39;28 called the DNA polymerase III holoenzyme,
00:09:42;19 which is a very specialized form of the DNA polymerase,
00:09:46;12 for acting at chromosomal DNA replication forks.
00:09:51;04 So, it's illustrated here,
00:09:53;11 and there are several parts to this complex.
00:09:56;06 So, first, there are three copies of
00:09:58;29 DNA polymerase III,
00:10:01;01 which is the third polymerase discovered by Arthur Kornberg and his colleagues
00:10:04;17 in their Nobel Prize-winning work investigating the enzymes involved
00:10:08;19 in DNA synthesis.
00:10:10;06 Now, in addition, there is
00:10:12;28 a second large protein complex,
00:10:15;16 a five-protein complex called a sliding DNA clamp loader,
00:10:17;16 as well as it being bound to a sliding DNA clamp.
00:10:22;14 Now, all of the polymerases
00:10:24;05 are held to the sliding clamp loader
00:10:26;04 by a subunit that's present three times,
00:10:28;23 shown here in light blue,
00:10:30;17 called the τ subunit,
00:10:32;10 and I'll have to say about τ,
00:10:34;22 as it plays a particularly important role in
00:10:37;11 coordinating the events at the replication fork.
00:10:39;19 But before that, I want to tell you a little bit about the sliding DNA clamp loader
00:10:42;15 and the sliding DNA clamp, and what their functions are.
00:10:46;12 So, we'll start with the sliding DNA clamp.
00:10:48;12 So, this is a ring-shaped multimeric protein
00:10:51;19 made up of either two or three identical subunits.
00:10:56;04 This is an illustration of the sliding DNA clamp
00:10:59;12 from S. cerevisiae, the budding yeast,
00:11:01;26 and you can see that whether we look at the one
00:11:04;09 from S. cerevisiae or a phage, T4,
00:11:06;08 or human cells, or E. coli,
00:11:10;20 they have very similar structures,
00:11:13;08 and you can see that in the overlap, here, okay?
00:11:15;03 And all of them, in this central
00:11:18;29 hole in the protein donut, per se,
00:11:20;14 have enough room to fit
00:11:23;19 double-stranded DNA.
00:11:24;26 And you can see that here in a crystal structure
00:11:27;01 of the yeast sliding DNA clamp
00:11:29;17 bound to double-stranded DNA.
00:11:32;01 Okay.
00:11:33;05 Now, what's the purpose of having it
00:11:35;08 surround double-stranded DNA.
00:11:36;22 Well, that's illustrated on the next slide.
00:11:40;16 So, these sliding DNA clamps
00:11:42;09 not only encircle double-stranded DNA,
00:11:43;21 but they also are able to
00:11:46;00 bind to the backside of a DNA polymerase,
00:11:49;24 holding it on the DNA,
00:11:51;13 particular at a primer-template junction, okay?
00:11:56;03 And it turns out, because they do not
00:11:58;20 specifically interact with the double-stranded DNA,
00:11:59;28 they will follow along with the polymerase
00:12:02;09 as it synthesizes DNA.
00:12:04;11 Now, one property we haven't talked about
00:12:06;16 of DNA polymerases thus far
00:12:08;12 is a property called processivity,
00:12:10;04 and this, put simply,
00:12:11;21 is the number of base pairs
00:12:13;20 that are synthesized
00:12:15;15 each time a DNA polymerase
00:12:17;16 binds to a primer-template junction.
00:12:19;13 Now, it turns out, on their own,
00:12:21;05 polymerases are actually not particular processive.
00:12:25;04 They'll typically do somewhere up to
00:12:28;07 about 100 base pairs
00:12:29;27 before they fall back off the DNA.
00:12:31;14 Now, what's important about this
00:12:33;25 is, while it stays on the DNA,
00:12:35;09 a DNA polymerase typically adds one base, one base pair
00:12:37;20 or makes one base pair, per millisecond,
00:12:42;13 and so it's very efficient at doing that
00:12:44;14 if it stays on the template.
00:12:46;06 However, if it falls back off the template,
00:12:48;15 on average,
00:12:50;11 it's going to take a second to find a new primer-template junction,
00:12:52;27 rebind, and reinitiate synthesis.
00:12:55;19 So, what that means is that every time it falls off,
00:12:58;23 it's lost the chance to add 1000 base pairs
00:13:01;05 if it stayed on.
00:13:02;28 And what the sliding clamp does
00:13:05;06 is prevent that.
00:13:06;21 In fact, I usually refer to these as
00:13:08;17 personal trainers for DNA polymerases,
00:13:10;18 because once it starts,
00:13:12;12 if the polymerase decides,
00:13:14;03 oh, I'm tired, I want to fall off,
00:13:15;25 the sliding clamp holds it on the DNA
00:13:18;01 and puts it right back to the grindstone,
00:13:20;17 and starts this process again.
00:13:23;04 And, importantly, it will only hold the polymerase
00:13:27;11 while active DNA synthesis is occurring.
00:13:29;26 When it reaches the end of a template
00:13:31;27 and you have complete synthesis,
00:13:33;23 it's readily released from the sliding clamp
00:13:35;18 and eventually the sliding clamp
00:13:37;15 is also removed from the DNA.
00:13:39;17 So, now that you know
00:13:42;02 the function of the sliding DNA clamp,
00:13:44;10 we need to talk about how it's put on
00:13:47;07 the primer-template junction
00:13:49;02 so that it can serve this function,
00:13:51;01 and this is the role of the so-called
00:13:52;20 sliding DNA clamp loaders.
00:13:54;26 So, these are five-subunit complexes
00:13:56;24 that use the energy of ATP binding and hydrolysis
00:14:00;02 to load sliding DNA clamps,
00:14:03;04 specifically at primer-template junctions.
00:14:06;04 Now, how does this work?
00:14:08;06 Well, the first step in this process
00:14:10;09 is the binding of ATP
00:14:12;16 by the sliding DNA clamp loader.
00:14:14;23 This changes its conformation
00:14:16;25 and makes it competent to bind both the sliding DNA clamp
00:14:19;16 and the primer-template junction.
00:14:22;10 When the sliding DNA clamp binds,
00:14:24;25 it changes its conformation
00:14:27;06 by opening up the interface
00:14:29;09 between two subunits,
00:14:31;14 creating a crack or an opening in the ring-shaped structure.
00:14:34;28 Importantly, this is big enough
00:14:37;17 to fit double-stranded DNA through,
00:14:39;06 and when double-stranded DNA
00:14:42;09 binds to the sliding DNA clamp loader,
00:14:43;16 it does so such that it is now encircled
00:14:47;11 by the sliding DNA clamp.
00:14:49;11 Importantly, only a DNA
00:14:52;00 that has a primer-template junction
00:14:54;02 can actually fit within this region.
00:14:56;15 Completely double-stranded DNA
00:14:58;20 can't bend enough to fit
00:15:01;02 in the sliding DNA clamp loading site.
00:15:04;06 Also important is the presence of
00:15:07;22 a 3' hydroxyl at the site of the ATP binding,
00:15:11;23 which stimulates the ability of ATP
00:15:14;25 to be hydrolyzed.
00:15:16;10 So, when ATP is hydrolyzed,
00:15:18;22 this causes the sliding clamp
00:15:21;19 to change conformations,
00:15:23;01 release the sliding clamp
00:15:25;00 and the primer-template junction DNA,
00:15:28;29 and this causes the sliding clamp
00:15:31;08 to now close again
00:15:32;28 around the double-stranded DNA portion
00:15:34;22 of the DNA,
00:15:36;16 and now it's ready
00:15:38;10 to recognize a DNA polymerase
00:15:39;27 and facilitate its processive DNA replication.
00:15:43;28 Now, at this point,
00:15:45;26 I've told you about a lot of different enzymes,
00:15:47;08 and I just want to tell you
00:15:49;09 a little bit about how they work together
00:15:51;00 at the replication fork.
00:15:52;25 So, the DNA polymerase III holoenzyme,
00:15:55;00 this large multi-enzyme complex,
00:15:57;04 actually does more than just synthesize DNA
00:16:00;24 and load the clamps
00:16:02;14 -- it also stimulates the DNA helicase.
00:16:04;24 And this is mediated, again,
00:16:06;12 by that same τ subunit
00:16:08;06 that is interacting with the DNA polymerase subunits,
00:16:11;11 and this plays an important role,
00:16:13;07 because if the DNA polymerase I,
00:16:15;26 either on the leading or lagging strand,
00:16:17;17 becomes stalled,
00:16:19;26 it will pull the sliding...
00:16:21;20 the Pol III holoenzyme away from the helicase,
00:16:24;07 causing the helicase to slow down
00:16:27;14 during the time it takes for the polymerase
00:16:29;12 to restart synthesis.
00:16:30;17 So, for example, if it hits a lesion in the DNA
00:16:32;09 that has to be repaired,
00:16:33;29 the helicase won't run away
00:16:35;29 at the same rate
00:16:37;24 as it would if it were bound to the polymerase,
00:16:39;18 because the polymerase is now detached.
00:16:41;11 Once the polymerase can bypass that lesion,
00:16:43;17 or the lesion is repaired,
00:16:44;29 it can catch back up to the helicase
00:16:46;27 and the process can become very rapid, again.
00:16:51;05 I've already told you that primase activity is stimulated
00:16:54;03 by binding to the DNA helicase,
00:16:55;22 and in fact if you modulate
00:16:57;29 the level of interaction, or the affinity,
00:17:00;12 of the primase for the helicase,
00:17:02;04 you can actually change the rate at which
00:17:04;14 it primes new syntheses,
00:17:06;05 and so if it's faster you'll make shorter Okazaki fragments,
00:17:09;04 and if it's slower, or lower affinity,
00:17:11;07 you'll make longer Okazaki fragments.
00:17:13;10 So, the rate of Okazaki fragment formation
00:17:15;14 is actually determined by this affinity.
00:17:19;10 Finally, DNA polymerase III, as I told you,
00:17:22;14 has its processivity dramatically stimulated
00:17:24;13 by the sliding clamp
00:17:26;14 and, in turn, also by the sliding clamp loader,
00:17:28;08 since that is required for loading it.
00:17:31;15 So, now that I've told you about the enzymes
00:17:34;10 and how they work together at the fork,
00:17:36;10 I'd like to take you through the events that are occurring
00:17:38;23 at a replication fork, one by one.
00:17:41;09 So, this is an illustration
00:17:43;20 of a replication fork
00:17:46;00 bound to the DNA polymerase III holoenzyme.
00:17:47;21 You can see the helicase, here,
00:17:49;12 with its unreplicated and unseparated DNA.
00:17:52;14 The top is the leading strand polymerase;
00:17:54;26 at the bottom is a lagging strand DNA polymerase.
00:17:57;22 There's also a third polymerase,
00:18:00;12 as I've explained,
00:18:01;29 which is, at this point in the reaction,
00:18:03;10 unengaged,
00:18:05;03 and you'll see how it becomes engaged as we move forward.
00:18:07;07 Now, you'll also note that
00:18:10;10 there's large regions of single-stranded DNA,
00:18:12;23 here and here,
00:18:14;06 that are bound to the single-stranded DNA binding protein.
00:18:17;00 And in fact, I've labeled this the trombone model
00:18:20;15 of bacterial DNA replication,
00:18:21;27 because this loop down here
00:18:24;06 actually gets bigger and smaller
00:18:26;07 depending on where you are in the replication process,
00:18:29;00 much as a trombone slide goes in and out
00:18:32;09 as you play different notes.
00:18:34;15 Now, I've shown you the SSB
00:18:36;25 that's bound to the single-stranded DNA, here,
00:18:38;17 but for the rest of the illustrations,
00:18:40;04 just to reduce the clutter,
00:18:42;10 I've removed that.
00:18:45;14 Now, you'll note that there's a large single-stranded region
00:18:47;20 adjacent to the helicase,
00:18:49;22 and this is actually the perfect substrate for the primase,
00:18:52;27 which will come in and synthesize
00:18:54;16 a short primer at this single-stranded DNA region.
00:18:57;10 And this is, again,
00:18:58;27 mediated by the affinity of the primer...
00:19:01;13 DNA primase for the helicase.
00:19:04;25 Now, as soon as this is synthesized,
00:19:06;22 it's recognized by the sliding DNA clamp loader
00:19:09;09 as a primer-template junction.
00:19:11;16 And it then loads a sliding DNA clamp
00:19:14;15 onto the primer-template junction,
00:19:16;14 making it, now,
00:19:18;18 ready to be recognized
00:19:21;01 by the unengaged DNA polymerase.
00:19:23;10 So, what happens next is that
00:19:24;27 the polymerase binds the sliding clamp,
00:19:27;07 associates with the primer-template junction,
00:19:30;07 and begins to initiate a second Okazaki fragment.
00:19:33;25 Now, I want to point out,
00:19:35;23 during this process...
00:19:37;18 the processes that I've been explaining,
00:19:39;02 the leading strand polymerase
00:19:40;23 has continued synthesizing,
00:19:42;11 as has the other lagging strand DNA polymerase.
00:19:45;12 And, in fact,
00:19:47;24 once that lagging strand polymerase
00:19:49;20 reaches the end of its single-stranded template
00:19:51;19 -- that is, the beginning of
00:19:54;01 the previous Okazaki fragment --
00:19:55;17 it will fall off the DNA, just as I explained,
00:19:59;21 because it no longer has template,
00:20:01;04 and it will become an unengaged DNA polymerase,
00:20:04;20 just as the one that is currently
00:20:07;05 making the second Okazaki fragment
00:20:09;07 was unengaged at the beginning of the reaction.
00:20:13;22 Okay. So, we've taken you through this in slow motion.
00:20:17;01 Let's look at what it looks like in real time.
00:20:20;23 Okay. So, this...
00:20:23;08 you should now be able to label
00:20:24;26 all these different subunits in this process.
00:20:27;04 So, you can see, in blue, here,
00:20:29;27 this is the DNA helicase, okay?
00:20:32;09 And it's unwinding the DNA, both strands,
00:20:35;03 and feeding it to two different DNA polymerases, here.
00:20:39;00 You can see the leading strand polymerase,
00:20:41;21 which is immediately using the template
00:20:46;10 on the leading strand...
00:20:48;06 using the leading strand template
00:20:49;24 to make new double-stranded DNA,
00:20:52;01 and you can see its associated
00:20:54;20 sliding DNA clamp, here, in the green.
00:20:57;05 Now, you'll also notice that
00:21:00;01 you see the arrival of a primase,
00:21:03;01 okay, here it comes, right there,
00:21:06;02 and it lays down a primer,
00:21:07;16 which is immediately recognized by the sliding DNA clamp loader,
00:21:09;29 which puts a new sliding DNA clamp on.
00:21:13;00 And in this case,
00:21:14;15 because the leading strand already has a sliding DNA clamp,
00:21:16;28 this is immediately recognized
00:21:19;04 by the lagging strand DNA polymerase, shown again in purple,
00:21:22;12 and its associated sliding DNA clamp.
00:21:25;17 Okay.
00:21:26;23 Now, it may be a little hard to...
00:21:28;15 oh, actually, I should point out...
00:21:29;22 there's one obvious difference
00:21:32;05 between the model I showed you and this model,
00:21:34;03 which is that there's only two DNA polymerases,
00:21:36;20 and that's because at the time this animation was made,
00:21:39;12 it wasn't actually known that there were three,
00:21:42;03 instead of two,
00:21:43;12 DNA polymerases.
00:21:44;25 But that turns out to be very important,
00:21:46;17 because, as you'll notice,
00:21:48;01 there's a period of time,
00:21:50;01 each time a new primer is synthesized,
00:21:52;05 and the lagging strand DNA polymerase
00:21:54;19 has to recognize that new template,
00:21:56;11 where there's no synthesis occurring
00:21:58;18 on the lagging strand template.
00:22:01;05 Now, in some organisms, this would be just fine,
00:22:03;00 but in E. coli the replication fork
00:22:05;09 actually moves at 1000 base pairs per second,
00:22:07;29 and as you'll recall that's the absolute maximum rate
00:22:11;23 a DNA polymerase can go.
00:22:15;11 So, while the leading strand polymerase can handle that quite easily,
00:22:17;07 the lagging strand polymerase couldn't do it
00:22:21;16 if it had to come off and rebind all the time.
00:22:22;27 On the other hand, if you have a third DNA polymerase,
00:22:26;06 it becomes obvious how you can always have
00:22:27;23 at least one polymerase, and often two,
00:22:29;23 acting on the lagging strand,
00:22:31;04 allowing the overall fork movement
00:22:33;03 to go at 1000 base pairs per second,
00:22:35;29 which is what's observed in vivo.
00:22:39;01 Now, sometimes it's hard to understand
00:22:41;02 how fast this really is,
00:22:42;16 so let me give you a little way to think about it
00:22:45;18 that's, I think, pretty impressive.
00:22:46;28 So, the double-stranded DNA
00:22:48;28 is 20 Angstroms wide,
00:22:50;13 but let's just imagine that it's 1 meter wide, okay?
00:22:54;02 Now, in that situation,
00:22:56;29 the nucleotides that would be incorporating
00:22:58;18 would be floating around the room you're in
00:23:01;20 at about the size of a textbook, okay?
00:23:03;07 And this replisome machine
00:23:05;10 would be about the size of a FedEx truck, okay?
00:23:08;18 The sliding DNA clamps would be the size of
00:23:11;08 very large wheels, okay?
00:23:13;05 And what's most impressive
00:23:15;28 for a replication fork moving at 1000 base pairs per second,
00:23:19;09 that FedEx truck, at the scale of the DNA
00:23:21;18 being a meter wide,
00:23:23;07 would be moving at 375 miles per hour.
00:23:26;21 So, if you were standing in a classroom
00:23:28;13 with a double hexamer tube
00:23:30;29 of 1 meter wide going across the classroom,
00:23:33;23 and let's say it's 50 meters...
00:23:35;21 50 feet across,
00:23:37;12 that replisome would come through,
00:23:39;20 spend about 0.1 second in the room,
00:23:41;26 and change that one tube of 1 meter into two tubes
00:23:47;06 before you would even really notice it. Okay?
00:23:48;28 So, these are really remarkable machines
00:23:50;13 that are accomplishing quite a bit at a time.
00:23:53;02 So, I want to end by
00:23:55;11 talking about a comparison
00:23:57;00 between the bacterial replication fork,
00:23:58;18 which I've told you about in great detail,
00:24:00;26 and what we know about the eukaryotic replication fork.
00:24:03;18 So, there are corresponding proteins
00:24:06;11 to all the proteins I talked about,
00:24:08;03 for the replication fork in bacteria,
00:24:10;25 found in eukaryotes.
00:24:13;04 However, it's a little bit more complicated there.
00:24:15;03 So, instead of the single
00:24:17;05 DNA polymerase III that's involved in replication at the eu... prokaryo...
00:24:21;24 bacterial replication fork,
00:24:23;12 there are actually three different DNA polymerases
00:24:25;10 that work at the eukaryotic replication fork.
00:24:27;15 Pol δ is exclusively making
00:24:29;19 the lagging strand DNA.
00:24:31;20 DNA Pol ε is exclusively making
00:24:34;09 the leading strand DNA.
00:24:35;24 Now, the third polymerase
00:24:38;24 is actually involved in the priming event.
00:24:40;12 So, while in E. coli the primase is
00:24:42;27 called DnaG and it's a single polypeptide,
00:24:44;20 in eukaryotic cells
00:24:46;04 it's a complex between a primase,
00:24:48;27 actually, a two-subunit primase,
00:24:50;26 and a DNA polymerase called
00:24:53;11 DNA polymerase α.
00:24:55;09 And the primase synthesizes
00:24:57;10 the short RNA primers
00:24:58;22 and then immediately hands them off to the DNA polymerase α,
00:25:02;28 which then extends them for a brief period of time
00:25:05;24 before, in turn,
00:25:07;09 allowing them to be taken over,
00:25:09;00 either by the Pol δ or the Pol ε,
00:25:12;04 leading or lagging strand polymerases.
00:25:14;24 As I showed you before, there's both E. coli
00:25:17;22 and eukaryotic sliding clamps.
00:25:20;02 In E. coli it's called β;
00:25:22;08 in eukaryotic cells it's called proliferating cell nuclear antigen,
00:25:25;01 or PCNA,
00:25:26;18 and, not surprisingly,
00:25:28;09 this was originally identified
00:25:29;26 just because it was prominently present
00:25:33;23 in dividing or proliferating cells,
00:25:35;10 thus the name PCNA.
00:25:36;19 The sliding clamp loader is called
00:25:39;12 the τ complex for reasons that you understand, now,
00:25:42;04 in bacteria,
00:25:43;17 and it's called the replication factor c, or RF-C,
00:25:46;12 in eukaryotic cells.
00:25:48;29 Again, the helicase is a little bit more complicated story
00:25:52;06 in eukaryotic cells versus bacterial cells.
00:25:55;09 It's a single subunit repeated six times,
00:25:58;19 the DnaB protein repeated six times,
00:26:02;08 in bacteria, but in eukaryotic cells the helicase,
00:26:04;26 the core of the helicase is the Mcm2-7 complex,
00:26:07;24 which is made of six different subunits
00:26:11;02 -- Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, and Mcm7 --
00:26:12;29 that form a hexamer with one of each subunit in the hexamer,
00:26:18;09 so it's a heterohexameric protein.
00:26:19;19 And it's not very active on its own.
00:26:21;09 Instead, it's activated
00:26:23;13 by binding to two other proteins,
00:26:24;26 Cdc45 and GINS,
00:26:26;28 to form the so-called CMG complex,
00:26:29;26 which is the active replicative helicase.
00:26:32;20 So, I've told you already that
00:26:35;02 there's three different DNA polymerases at the eukaryotic fork.
00:26:37;18 We know less about the interactions at the fork as well,
00:26:40;23 although we do know that Pol ε
00:26:42;21 is held at the replication fork
00:26:44;14 by interactions with the GINS protein.
00:26:46;06 In contrast,
00:26:48;02 neither Pol δ nor the eukaryotic clamp loader
00:26:50;15 is part of the replication fork,
00:26:52;06 so that's very different from the prokaryotic fork.
00:26:55;06 And this may be because
00:26:57;19 eukaryotic forks move at a much more leisurely pace
00:27:00;13 of 20-60 base pairs per second,
00:27:02;14 compared to the 1000 base pairs per second,
00:27:05;01 and so those lagging strand events
00:27:07;06 that have to be tightly coordinated in bacteria
00:27:10;05 can, quite likely,
00:27:13;10 occur by just solution binding out of...
00:27:16;12 instead of being tethered immediately
00:27:19;25 to the site of DNA replication,
00:27:21;04 because there's much more time for them to occur.
00:27:24;03 And, in fact, for lagging strand synthesis,
00:27:27;16 it probably occurs after the fork has gone by.
00:27:31;12 So, at this point I hope you understand
00:27:33;10 a lot more about how the replication fork works
00:27:36;04 and, if you stay tuned to my next presentation,
00:27:39;07 we'll talk about how you assemble these replisomes
00:27:42;11 at sites of initiation
00:27:44;04 and understanding how that's regulated during the cell cycle.
00:27:48;26 So, I just want to thank the people involved in this process.
00:27:51;20 So, Sera Thornton
00:27:53;25 did most of the animations that you saw.
00:27:55;08 They were done in the MITx Biology office
00:27:57;26 as part of the development of a course
00:28:00;29 that Sera and Mary Ellen Wiltrout
00:28:03;26 helped me develop called 728x,
00:28:06;00 which is available on the MITx and edX platforms,
00:28:11;05 and is a full molecular biology course,
00:28:13;15 talking about all the elements of the central dogma.
00:28:16;07 In addition, the very high resolution animation
00:28:19;19 showing the E. coli replication fork in action
00:28:22;22 was done in the DNA Learning Center,
00:28:25;07 which is part of the Cold Spring Harbor Laboratory.
00:28:28;13 Finally, I want to thank
00:28:30;25 the Howard Hughes Medical Institute
00:28:32;12 and the National Institute of General Medical Sciences
00:28:34;04 for supporting my research.

Part 2: Mechanisms of Chromosomal DNA Replication: Initiation of DNA Replication

00:00:08;09 Hi. My name is Steve Bell,
00:00:09;15 and I'm a professor of biology at MIT
00:00:11;23 and an investigator of the Howard Hughes Medical Institute.
00:00:14;22 And what I'd like to tell you about now
00:00:16;12 is the mechanisms controlling
00:00:20;19 the initiation of DNA replication at chromosomes.
00:00:22;10 This is a continuation of my previous discussion
00:00:25;28 of the events that occur at the replication fork.
00:00:32;01 Now, this process involves two key events.
00:00:33;19 First, unwind part of the DNA
00:00:37;03 to make single-stranded templates
00:00:39;11 to assemble the replisome on.
00:00:40;28 And, secondly, you need to
00:00:43;00 assemble two copies of the replisome,
00:00:44;26 because all genomic
00:00:48;05 or chromosomal origins of replication
00:00:49;24 initiate DNA replication bidirectionally,
00:00:53;00 meaning that they...
00:00:54;06 the replisomes that are assembled
00:00:57;07 move away in both directions
00:00:59;23 from the starting site of replication.
00:01:01;16 So, the start sites of DNA replication
00:01:03;04 are called origins of replication,
00:01:06;13 and this is where the first single-stranded DNA is formed
00:01:08;08 and where the first DNA synthesis occurs.
00:01:09;23 They can fire with various so-called efficiencies,
00:01:13;07 which is defined as the percentage of cell divisions
00:01:15;28 in which an origin initiates DNA replication.
00:01:19;00 Some origins have high efficiencies,
00:01:21;25 others have lower,
00:01:23;06 and I'll tell you more about that in a moment.
00:01:25;16 Now, all the DNA that's replicated
00:01:28;04 by the two replisomes that are formed
00:01:29;28 at each origin of replication...
00:01:31;17 and, a reminder,
00:01:33;13 all chromosomal origins are bidirectional...
00:01:35;05 that DNA is called a replicon.
00:01:37;21 And this stems from a model proposed in 1963
00:01:40;25 by Jacob, Brenner, and Cuzin
00:01:42;18 for the mechanism of initiation
00:01:45;01 of bacterial replication.
00:01:47;04 It's a very simple model.
00:01:48;24 They proposed only two components:
00:01:50;17 the replicator and the initiator.
00:01:52;21 The replicator is the DNA sequences
00:01:55;20 that are required to initiate DNA replication.
00:01:58;07 The initiator is a protein
00:02:00;21 that was proposed to bind to the replicator
00:02:02;24 and trigger the initiation of DNA replication.
00:02:06;24 Now, the replicator,
00:02:09;06 although it typically overlaps the origin of replication,
00:02:10;24 is not the same as that,
00:02:12;25 and it is instead all the sequences
00:02:15;12 that are required.
00:02:16;11 And this is sometimes a little bit difficult to understand,
00:02:18;10 but think about it in terms of transcription,
00:02:20;27 where the promoter is all the sequences
00:02:23;29 required to correctly regulate initiation of transcription
00:02:27;07 from the gene,
00:02:28;18 but the transcriptional start site
00:02:30;08 is a very specific sequence
00:02:32;11 at which initiation actually occurs.
00:02:34;06 In that analogy,
00:02:35;24 the promoter is equivalent to the replicator,
00:02:38;05 and the transcriptional start site
00:02:39;22 is equivalent to the origin of replication.
00:02:43;08 So, this is what origins are like
00:02:47;20 and what replicators are like...
00:02:49;15 how are they distributed across the chromosomes?
00:02:51;12 So, in E. coli,
00:02:55;00 not surprisingly, where there's a single origin of replication,
00:02:56;13 the origins are 100% efficient.
00:02:58;22 If an origin doesn't fire,
00:03:00;18 that means one of the two daughter cells
00:03:02;02 is not getting a whole chromosome.
00:03:05;13 This is different in eukaryotic cells,
00:03:07;13 where there are many, many origins.
00:03:09;03 What's found is that most origins
00:03:11;10 do not initiate 100% of the time,
00:03:13;07 but that's okay,
00:03:15;01 because when they don't initiate,
00:03:16;24 the replication forks on either side
00:03:19;01 will replicate through the region
00:03:20;29 that they would have replicated otherwise,
00:03:23;00 because there's lot of excess replication capacity.
00:03:26;29 It's important to note that
00:03:28;26 any origin that doesn't initiate
00:03:30;11 but is replicated
00:03:31;23 is inactivated.
00:03:33;08 So, you never have an origin initiate
00:03:35;12 after it's been replicated by an adjacent fork.
00:03:38;17 Now, another interesting characteristic of eukaryotic replication
00:03:42;00 is that when origins initiate
00:03:45;06 is characteristic of different origins.
00:03:46;12 Some will initiate early during the S phase of the cell cycle,
00:03:49;29 the time in the eukaryotic cell cycle
00:03:52;21 when DNA replication occurs,
00:03:54;20 others will replicate in the middle,
00:03:56;00 and still others will replicate late in S phase.
00:03:58;24 And it's sort of initially unclear
00:04:02;03 why you'd want to distribute this.
00:04:03;16 Why not replicate everything all at once?
00:04:05;22 But it's more clear when you think about the fact
00:04:08;29 that sometimes you'll have damage to your DNA
00:04:11;09 -- this could occur by
00:04:13;07 a sudden change in your environment --
00:04:17;07 and if you do and you stop two forks,
00:04:19;00 and they haven't replicated the intervening DNA,
00:04:22;28 the only way to rescue that situation
00:04:25;10 is to initiate replication
00:04:27;09 between those two forks
00:04:28;25 and create two new replisomes
00:04:30;11 that can fill in that gap.
00:04:31;26 And by reserving a subset of origins
00:04:34;10 to initiate late in S phase,
00:04:36;10 you will increase the likelihood
00:04:38;26 that you can fix a gap
00:04:40;27 that's caused by some sort of DNA damage
00:04:42;25 and stalling of replication forks.
00:04:45;26 Okay?
00:04:47;24 So, we've talked about origins,
00:04:50;24 let's talk about how initiation occurs?
00:04:52;10 So, we're going to start with the initiation of DNA replication in bacteria.
00:04:55;01 So, this is an illustration of the E. coli origin of replication.
00:04:59;13 It has 5 copies
00:05:01;23 of the so-called 9-mer repeats,
00:05:03;04 which are the binding sites
00:05:05;08 for the bacterial initiator protein,
00:05:06;21 which is called DnaA,
00:05:08;14 and 3 copies of the 13-mer repeat,
00:05:11;03 which is an AT-rich,
00:05:12;26 easily unwound DNA sequence.
00:05:15;26 Now, when the DnaA is present,
00:05:20;14 which is... and active in the cell,
00:05:22;00 and this is a highly regulated step
00:05:24;03 in the process in bacterial cells,
00:05:26;24 it will bind to the 9-mer sequences.
00:05:31;10 This is turn forms a helical filament,
00:05:34;14 which distorts the DNA,
00:05:36;19 causing the DNA it's bound
00:05:39;13 to have positive superhelicity,
00:05:40;28 and the adjacent DNA to, in a compensatory fashion,
00:05:44;28 generate negative superhelicity,
00:05:46;15 which favors unwinding
00:05:49;06 of the adjacent 13-mer regions.
00:05:51;26 Interestingly,
00:05:53;20 the DnaA that forms this filament
00:05:55;19 can actually extend that filament
00:05:57;24 by binding the single-stranded DNA
00:05:59;18 rather than the double-stranded DNA,
00:06:01;23 although it only does this on the top strand,
00:06:04;08 as illustrated here,
00:06:06;03 not on both strands.
00:06:08;07 Now, at this point,
00:06:10;03 we've accomplished one important event,
00:06:11;08 which is to unwind the DNA at the origin,
00:06:13;29 but we're clearly lacking
00:06:15;28 much of the rest of the machinery,
00:06:17;13 and the next thing we need to initiate replication
00:06:20;20 is to load the replicative helicase...
00:06:23;01 with DnaB helicase,
00:06:25;07 the replicative helicase.
00:06:26;26 Now, it turns out that it doesn't arrive at the origin on its own;
00:06:30;29 it's bound to another protein, DnaC,
00:06:33;00 which is a so-called helicase loader,
00:06:35;04 and it has many important properties
00:06:37;09 that make it ideal for loading the helicase on the DNA.
00:06:41;22 So, one property is when DnaC is bound to DnaB,
00:06:46;04 it inactivates its helicase activity,
00:06:47;10 preventing it from acting at non-origin regions of the genome.
00:06:51;19 Secondly, DnaC causes the DnaB ring
00:06:55;13 to crack open such that it has
00:06:58;17 enough room to fit
00:07:01;02 a single-stranded DNA
00:07:04;03 so that it can gain access into
00:07:05;29 the central channel of the hexameric ring structure.
00:07:10;09 The third important property is that
00:07:12;21 DnaC has affinity for DnaA,
00:07:15;08 which recruits the DnaB-C complex
00:07:18;16 to the single-stranded DNA
00:07:20;23 and allows it to now
00:07:23;17 encircle the DNA,
00:07:25;04 so it binds such that it will now
00:07:27;05 encircle the single-stranded DNA.
00:07:29;04 Now, importantly,
00:07:31;00 not only does the DnaB-C complex
00:07:33;05 load on the top strand
00:07:35;13 that has DnaA associated with it,
00:07:37;16 but it also is loaded on the bottom strand,
00:07:39;08 but in the opposite orientation,
00:07:40;26 so that it will leave the replication...
00:07:43;14 the origin in the opposite direction.
00:07:46;03 So, you can see that on the top
00:07:48;02 it's gonna move to the left,
00:07:49;19 on the bottom it's gonna move to the right.
00:07:53;04 Now, we don't understand in detail
00:07:55;08 how that second DnaB-C complex is recruited,
00:07:58;08 although there are theories that suggest that
00:08:00;26 DnaA, which also has affinity for DnaB,
00:08:04;11 may use that interaction
00:08:06;17 to recruit the second B-C complex
00:08:08;05 in the opposite orientation.
00:08:10;25 Now, at this point,
00:08:12;03 you'll remember that the B-C complex is inactive,
00:08:15;05 so the helicase is not active yet.
00:08:17;25 Nevertheless, you'll recall that the
00:08:21;05 interaction between the DNA primase
00:08:23;02 and the helicase
00:08:24;18 stimulates the primase to synthesize new primers,
00:08:27;02 and that's the next step of this process.
00:08:29;11 The DNA primase is recruited,
00:08:32;02 binds to the helicase,
00:08:33;18 and synthesizes a primer,
00:08:35;20 and something about this process
00:08:38;00 causes the DnaC subunits to release.
00:08:40;25 Again, we don't understand that in detail,
00:08:43;04 but that's clearly the step at which this occurs,
00:08:45;07 and now you have active helicases,
00:08:47;08 which can move to the right and the left
00:08:50;07 -- top to the left, bottom to the right --
00:08:52;14 to unwind more DNA,
00:08:54;09 so that you get an extended unwound region.
00:08:57;21 Now, at this point, we have two primer-template junctions
00:09:01;11 that can be recognized by DNA polymerase III holoenzyme,
00:09:04;06 load sliding clamps,
00:09:06;17 and then the sliding clamps can be recognized
00:09:08;07 by one of the three DNA polymerase III enzymes.
00:09:12;06 It will then initiate leading strand synthesis.
00:09:15;14 Then, at some rate determined by the affinity
00:09:18;22 between the helicase and the primase,
00:09:20;20 a second primer will be synthesized.
00:09:22;23 That will be recognized by the clamp loader
00:09:24;28 and a second polymerase subunit,
00:09:27;07 and then you have initiated
00:09:29;13 both leading and lagging strand synthesis,
00:09:31;16 and this is really the starting point
00:09:33;06 that we discussed in terms of the function
00:09:35;24 of the replisome during elongation,
00:09:37;23 so you know what happens from here.
00:09:40;15 Now, I want to make a couple other points
00:09:43;07 at this point.
00:09:44;03 You'll note that there was only a single
00:09:47;22 sequence-specific DNA binding protein involved in this process.
00:09:49;09 Most of the process is driven
00:09:52;09 by either protein-protein interactions
00:09:54;18 or non-specific
00:09:56;29 or structure-specific protein-DNA interactions.
00:09:59;05 So, for example,
00:10:01;07 the recognition of the primer-template junction
00:10:02;29 is sequence-non-specific,
00:10:04;19 but very specific for the particular structure,
00:10:08;00 that of a primer with the 3' OH available
00:10:11;18 and an adjacent single-stranded region
00:10:13;24 for the DNA polymerase to be engaged with.
00:10:17;07 Okay?
00:10:18;08 And that's true also for the primase
00:10:20;06 interacting with the helicase
00:10:22;03 and the DNA polymerase III holoenzyme
00:10:24;19 interacting with the helicase as well.
00:10:27;07 So, this is built not by
00:10:29;17 a series of sequence-specific interactions,
00:10:31;13 but by a series of
00:10:33;24 protein-protein-specific interactions
00:10:35;15 or structure-specific interactions.
00:10:38;27 Okay, so that's the initiation in bacteria.
00:10:41;08 I want to turn my attention, now,
00:10:43;23 to initiation in eukaryotic cells,
00:10:46;04 which is substantially more complex.
00:10:49;28 So, this is just an overview of the events
00:10:52;16 of eukaryotic DNA replication,
00:10:54;07 and I first want to discuss the overview,
00:10:56;08 then I'm going to describe
00:10:58;10 how these events are regulated,
00:11:00;03 and then I'm going to take you through the detailed steps
00:11:02;18 of this initiation event.
00:11:05;05 So, the first step in this process
00:11:07;10 occurs not during the S phase of the cell cycle,
00:11:08;29 when DNA synthesis occurs,
00:11:10;21 but during the G1 phase of the cell cycle,
00:11:13;11 so the preparations for replication occur
00:11:15;29 well before any nucleotide is added
00:11:19;01 to any DNA strand.
00:11:21;27 During this helicase loading process in G1,
00:11:26;04 the replicative helicase, this Mcm2-7 complex,
00:11:29;01 is assembled around the DNA at each origin.
00:11:33;00 In fact, this marks all potential origins in cells
00:11:37;01 and is frequently referred to as origin licensing
00:11:39;11 or sometimes pre-replicative complex formation.
00:11:43;13 Three additional proteins are required to accomplish this.
00:11:47;25 In addition to the helicase,
00:11:49;10 the origin recognition complex,
00:11:51;20 Cdc6,
00:11:53;05 and Cdt1,
00:11:55;01 and I'll have much more to say about them in a few minutes.
00:11:58;17 As the cells go from the G1 phase
00:12:01;04 to the S phase of the cell cycle,
00:12:02;24 the action of two kinases,
00:12:05;03 the S phase cyclin-dependent kinase,
00:12:07;01 and the Dbf4-dependent kinase, or DDK,
00:12:10;21 leads to the accumulation of a number of different proteins
00:12:13;28 onto the loaded replicative helicase,
00:12:16;28 because what I didn't tell you is that
00:12:19;23 when it's initially loaded
00:12:21;14 it's surrounding double-stranded DNA
00:12:23;11 and it's completely inactive, okay?
00:12:25;26 So, it's important that it's inactive at that point
00:12:27;20 and you'll see why.
00:12:28;28 Now, these associated proteins
00:12:30;29 include two proteins that I discussed before,
00:12:33;15 Cdc45 and GINS,
00:12:35;27 and they come together
00:12:38;18 to activate the helicase and
00:12:41;04 form the active replicative helicase,
00:12:42;23 the CMG complex.
00:12:44;04 Now, the single-stranded DNA
00:12:46;12 that's formed at this point
00:12:48;02 allows the recruitment of the rest of the DNA synthetic machinery
00:12:51;00 and assembly of a pair of bidirectional replisomes.
00:12:55;04 Now, what's important about this process
00:12:56;29 is that the helicase loading step
00:12:59;17 is separated from the helicase activation step.
00:13:02;20 And this is critical for one
00:13:04;27 very important attribute of eukaryotic DNA replication,
00:13:07;27 which is that the genome is replicated
00:13:10;09 exactly once per cell cycle.
00:13:12;16 You never want an origin to initiate
00:13:14;24 more than once per cell cycle.
00:13:16;22 So, I want to talk about how the cell ensures that
00:13:19;10 over the next couple of slides.
00:13:21;12 So, during the G1 phase of the cell cycle,
00:13:23;13 there is low levels of an enzyme
00:13:26;19 called cyclin-dependent kinase.
00:13:28;19 This is the key enzyme
00:13:30;21 that drives cell cycle progression.
00:13:32;03 During G1, there's only
00:13:34;20 G1 cyclin-dependent kinases
00:13:36;07 -- no S phase cyclin-dependent kinases --
00:13:38;15 and in general their activity is much lower.
00:13:41;07 Under these conditions,
00:13:43;18 you can assemble or load new replicative helicases
00:13:46;19 at the origins of replication,
00:13:48;00 but you cannot activate them, because,
00:13:50;05 as I told you in the previous slide,
00:13:51;22 you require S phase CDK activity for this.
00:13:55;22 When you transition into the S phase of the cell cycle,
00:14:00;01 S phase CDKs,
00:14:01;28 and in fact CDKs in general,
00:14:03;14 become much more represented,
00:14:05;23 much higher activity,
00:14:07;05 and they have two consequences.
00:14:09;02 One is that they
00:14:12;17 trigger initiation of any loaded helicases,
00:14:15;18 as I described in the previous slide.
00:14:17;29 The second important consequence
00:14:19;27 is that they also target
00:14:22;12 three of the four proteins
00:14:24;21 involved in helicase loading
00:14:26;24 -- the Mcm2-7 complex, ORC, and Cdc6 --
00:14:31;13 and each of these phosphorylation events
00:14:33;27 inhibits helicase loading.
00:14:36;06 I'm going to describe that in detail in a moment,
00:14:38;13 but let me just explain the logic of this system first.
00:14:41;21 So, you can see that in this system
00:14:43;29 there's only one chance, during G1,
00:14:45;25 to load a replicative helicase per cell cycle,
00:14:48;17 and there's only one chance,
00:14:50;14 during S phase, typically,
00:14:52;02 but really the whole period from S, G2, and M,
00:14:54;26 where you could actually activate a helicase.
00:14:57;00 In practice, this all occurs during S phase
00:15:00;05 for a variety of reasons that I won't go into,
00:15:02;27 but what this means is
00:15:05;10 you basically can only initiate replication from an origin
00:15:08;00 once per cell cycle.
00:15:09;28 And this system works rather you have
00:15:11;18 10 origins, 1000 origins, or 30000 origins.
00:15:15;05 It's going to work equally well.
00:15:17;04 And so it's very able to accommodate
00:15:19;09 many different genome sizes.
00:15:21;10 Now, you might wonder what happens
00:15:23;13 at the G1-S and M-G1 transitions.
00:15:26;13 That's really a dangerous period
00:15:28;15 where you want to stop helicase loading
00:15:31;00 before you start helicase activation.
00:15:32;26 If there was some gradual transition,
00:15:34;28 you could have a brief period
00:15:36;28 where they both could occur at the same time,
00:15:38;15 and that could allow re-replication to occur.
00:15:41;11 However, it turns out the cell cycle
00:15:43;16 has come up with mechanisms,
00:15:45;25 and I won't go through these in detail,
00:15:47;12 that turn off helicase loading
00:15:49;14 before you turn on helicase activation
00:15:52;00 at the G1-S transition,
00:15:53;25 and similarly you turn off activation
00:15:56;13 before you turn on helicase loading
00:15:59;29 at the M-G1 transition.
00:16:01;13 So, at each of these key transitions,
00:16:03;10 you have a little window
00:16:05;26 where neither of them can occur.
00:16:07;29 Okay?
00:16:09;27 And I should just emphasize that this transition
00:16:11;22 at the M to G1
00:16:13;22 is exactly where cell division occurs,
00:16:15;10 so you can now start the process again,
00:16:18;17 because you now have two cells,
00:16:20;04 each of which has a complete complement
00:16:22;13 of the genome in it
00:16:24;06 and is ready to initiate a new round
00:16:26;26 of cell division and therefore DNA synthesis.
00:16:30;19 Now, how does S CDK
00:16:33;05 control the helicase loading proteins?
00:16:35;01 We know that best in the budding yeast S. cerevisiae,
00:16:37;24 where we know that, for example,
00:16:40;06 cyclin-CDKs phosphorylate the Mcm proteins,
00:16:43;05 which causes them to be
00:16:45;17 exported out of the nucleus.
00:16:47;00 Now, this may be confusing to you
00:16:48;26 because obviously there are many Mcm proteins
00:16:51;28 that are loaded onto the DNA
00:16:53;15 that you don't want to leave the nucleus,
00:16:55;13 and the important point here is that
00:16:57;03 only non-DNA associated Mcms
00:17:00;11 are exported.
00:17:02;03 So, as soon as they finish their function,
00:17:03;16 they're exported to the cytoplasm,
00:17:04;23 but if they're waiting at an origin, loaded,
00:17:07;07 to be activated,
00:17:08;24 they are not exported out by CDK phosphorylation.
00:17:12;17 Cdc6 is also phosphorylated by CDK,
00:17:16;04 and that phosphorylation
00:17:18;00 leads to its recognition by a protein complex called SCF,
00:17:21;24 which is a ubiquitin ligase,
00:17:23;22 which targets Cdc6
00:17:26;25 with multiple modifications of ubiquitin,
00:17:28;18 which in turn causes Cdc6
00:17:31;27 to be recognized by the proteasome and degraded.
00:17:35;02 So, except for in G1,
00:17:36;20 Cdc6 is consistently being degraded
00:17:38;10 and therefore cannot function.
00:17:40;14 Finally, ORC is also phosphorylated,
00:17:42;22 and in this case we know less about this,
00:17:44;25 but it's prob...
00:17:46;26 most likely an event that inhibits the interaction of
00:17:50;14 one of the other helicase loading proteins
00:17:52;06 with ORC.
00:17:54;01 Okay.
00:17:55;22 So, I've told you about the regulation of this event,
00:17:57;19 let's talk about how it actually occurs.
00:18:00;17 So, this is just an illustration of the events of helicase loading.
00:18:04;10 We know that the first step in this process
00:18:06;22 is the recognition of the origin DNA
00:18:08;20 by the origin recognition complex,
00:18:10;06 or what I'll call ORC from now on.
00:18:12;25 ORC then recruits Cdc6
00:18:15;22 as cells enter into the G1 phase of the cell cycle,
00:18:17;13 when it's newly synthesized,
00:18:19;13 and then the ORC-Cdc6 complex
00:18:22;05 can recruit a complex between the Mcm2-7 helicase and Cdt1.
00:18:28;07 And this forms this initial complex
00:18:30;05 called the OCCM,
00:18:31;29 for ORC, Cdc6, Cdt1, Mcm complex.
00:18:34;22 And I want to point out that, at this point,
00:18:38;25 it's the C-terminal end of the Mcm complexes,
00:18:40;22 right here,
00:18:42;09 that's interacting with ORC.
00:18:44;01 The ring is still open at the this point,
00:18:45;27 but they're bound to adjacent pieces of DNA,
00:18:48;09 making it very clear how you recruit
00:18:50;29 the first Mcm to the DNA.
00:18:53;20 Now, the next step after this
00:18:56;29 requires ATP hydrolysis,
00:18:58;17 and involves a number of steps
00:19:00;15 that I'll talk about in more detail
00:19:02;15 in my second part of my presentation.
00:19:04;16 But the end result is that
00:19:07;15 you end up with a double hexamer of Mcms on the DNA,
00:19:10;02 with then interacting
00:19:12;22 via their N-terminal regions
00:19:14;24 to form a head-to-head double hexamer
00:19:17;06 of the proteins on the DNA,
00:19:18;18 and this is really the first marker of bidirectional initiation,
00:19:22;00 because they are now poised
00:19:24;11 to leave the origin in opposite directions.
00:19:28;02 So, this happens during G1.
00:19:30;29 Again, the helicases are inactive at this point,
00:19:33;10 and what happens next is you
00:19:35;27 transition into S phase
00:19:38;07 and you undergo helicase activation.
00:19:39;21 Now, this is an overview of this process,
00:19:41;21 and I'm going to go over this first
00:19:43;28 and then I'm going to go over the steps in detail.
00:19:46;15 So, the first event is...
00:19:48;12 it requires the DDK kinase
00:19:50;25 and it results in the recruitment
00:19:53;00 of one of the two activators,
00:19:54;29 the Cdc45 protein.
00:19:57;26 Then, S-CDK can act
00:19:59;28 and this recruits the other activator,
00:20:02;19 the GINS complex,
00:20:04;11 as well as Pol ε and several other proteins.
00:20:07;17 At this point,
00:20:10;24 you have two CMGs that have formed on the template,
00:20:13;11 but interestingly they are not yet active.
00:20:16;05 It requires the activity of a third protein,
00:20:18;16 the Mcm10 protein,
00:20:20;26 to actually trigger DNA unwinding
00:20:22;28 and CMG activation.
00:20:25;00 And this also requires the association
00:20:27;11 of the eukaryotic single-stranded binding protein
00:20:30;00 RPA.
00:20:32;01 Finally, once the single-stranded DNA is formed,
00:20:34;02 the remaining DNA polymerases,
00:20:37;08 as well as other auxiliary proteins,
00:20:39;08 are recruited to form the replisome.
00:20:41;28 Now, this is a lot to look at,
00:20:44;09 so I want to break this down into individual steps
00:20:46;29 so you can understand how this occurs in more detail.
00:20:50;08 So, here's our loaded double hexamer,
00:20:52;18 and it's ready to be activated,
00:20:55;06 just entering into S phase.
00:20:57;23 So, the first step is the DDK recognition
00:21:00;20 of the N-terminal region
00:21:02;19 of several of the Mcm subunits.
00:21:05;13 It phosphorylates these subunits
00:21:07;16 and that creates a binding site
00:21:09;21 for a second protein called Sld3,
00:21:13;12 which in turn allows the recruitment
00:21:15;10 of one of the two activating proteins,
00:21:18;01 Cdc45.
00:21:19;18 Now, I've shown this for the right hexamer,
00:21:21;19 but of course this also has to happen
00:21:23;14 for the left hexamer as well.
00:21:26;06 Okay, so now we have the first activator present,
00:21:30;12 and that's a DDK, or Dbf4-dependent kinase-dependent event.
00:21:33;11 The next step absolutely requires
00:21:36;17 the S phase cyclin-dependent kinase,
00:21:38;10 and what it does it to
00:21:41;14 phosphorylate, first, Sld3,
00:21:44;05 and then, second, it phosphorylates...
00:21:47;06 not necessarily in that order,
00:21:48;25 but it also phosphorylates Sld2.
00:21:51;17 Now, these phosphorylation events
00:21:53;08 cause the recruitment of a variety of additional proteins.
00:21:56;18 Most notably,
00:21:58;24 Sld2 is recognized by a protein called Dpb11,
00:22:01;16 and it does this using a pair of BRCT repeat motifs,
00:22:06;07 which recognize,
00:22:08;00 in a phosphorylation-specific manner,
00:22:09;16 a peptide on Sld2.
00:22:11;23 This phosphorylation event
00:22:14;03 also causes the recruitment of
00:22:16;18 Pol ε and the GINS protein
00:22:19;04 into a larger complex
00:22:21;20 that's called the pre-landing complex, or pre-LC.
00:22:25;19 Now, this complex is recruited as a whole
00:22:29;00 to the inactive,
00:22:32;06 ready-to-be-activated helicase,
00:22:34;24 using the interaction of the Dpb11 protein
00:22:38;20 with a different phosphorylation...
00:22:41;00 phosphorylated peptide, okay?
00:22:43;17 And this, again, is mediated by
00:22:46;03 a separate pair of BRCT motifs,
00:22:48;16 which recognize,
00:22:50;17 in a phosphorylation-specific manner,
00:22:52;06 peptides within Sld3.
00:22:54;23 So, at this point, we've recruited
00:22:57;06 both Cdc45 and GINS,
00:22:59;08 and so we have
00:23:01;29 a CMG complex present,
00:23:03;19 okay, but it's not active yet.
00:23:05;03 And of course we also have to do this
00:23:07;27 for the other hexamer.
00:23:09;08 Okay, so what happens next
00:23:12;04 is really a point of major investigation in the field,
00:23:16;04 but there are a number of things we know must happen.
00:23:18;22 So, one of these is that
00:23:20;21 several of the proteins leave the replisome.
00:23:23;09 This includes Sld2, Sld3, and Dpb11.
00:23:27;20 So, we have our CMG complex,
00:23:29;13 and this is, again, the active replicative helicase,
00:23:32;04 but it's not active yet
00:23:33;29 because in order for the first unwinding of DNA to occur
00:23:37;28 the Mcm10 protein has to act.
00:23:40;20 And we know that this actually
00:23:43;07 directly stimulates helicase activity,
00:23:45;12 and that it can
00:23:47;18 cause a number of changes
00:23:49;21 in order to cause initial unwinding.
00:23:52;11 Exactly the order of these changes,
00:23:55;15 and whether Mcm10 or earlier events may stimulate them,
00:23:58;03 is unknown.
00:23:59;12 We only know that you don't see single-stranded DNA
00:24:01;07 until Mcm10 arrives.
00:24:03;25 But this structure,
00:24:05;28 which has now got RPA
00:24:08;14 binding to the single-stranded regions,
00:24:10;01 has a number of dramatic changes
00:24:12;00 versus the previous structure.
00:24:13;20 So, of course the origin DNA
00:24:15;09 had to be melted in order to create
00:24:17;24 single-stranded DNA.
00:24:18;28 In addition, the non-translocating strand
00:24:22;15 had to be exported from the central channel.
00:24:26;03 Remember, the Mcms were around
00:24:28;04 double-stranded DNA initially,
00:24:29;16 however, after this transition
00:24:31;22 the right helicase
00:24:34;00 is going to have one single-strand present in it,
00:24:36;18 and the left helicase
00:24:38;20 is going to have a different single-strand in it.
00:24:40;10 So, that means these Mcm complexes
00:24:42;10 have to open, eject one single-strand,
00:24:44;22 and then close again around another.
00:24:47;15 And, finally, you'll recall that
00:24:49;22 the helicases are in this tight, head-to-head double hexamer, initially,
00:24:53;04 and they have to separate in this process.
00:24:55;04 So, I've listed these in an order,
00:24:57;12 but we don't know if this is actually the order
00:24:59;24 they occur in
00:25:01;16 -- that's an area of active investigation.
00:25:03;23 Okay.
00:25:05;19 So, at this point we know have the single-stranded DNA
00:25:07;22 necessary for priming,
00:25:09;18 and what happens is that
00:25:11;25 the rest of the DNA synthetic machinery is recruited.
00:25:14;22 In particular, Pol α-primase,
00:25:16;23 is recruited by interactions of
00:25:19;21 Ctf4 with the GINS complex,
00:25:23;16 and also with Pol α.
00:25:25;15 This allows the synthesis of the first RNA primers
00:25:30;07 and then the extension of those primers by Pol α in the form of DNA.
00:25:33;27 Those primers can then be taken over,
00:25:36;12 either by Pol δ or by Pol ε,
00:25:39;12 depending on whether they're on the leading
00:25:41;16 or the lagging strand,
00:25:43;00 and you now have an active replisome.
00:25:45;23 So, there's lots more to be understood
00:25:49;02 about this process
00:25:50;21 -- it's much less well-understood
00:25:52;14 what the architecture of this complex is --
00:25:54;07 and there's a lot of investigation going on at this point
00:25:57;03 to understand that.
00:25:58;08 For example, I've illustrated Pol δ
00:26:01;01 being at the site of replication,
00:26:02;26 but a lot of evidence suggests that it's actually after...
00:26:07;00 it's occurring sort of after the fork has gone by.
00:26:08;24 So, it's doing its job,
00:26:10;13 but somewhat separately from the job
00:26:12;16 of the rest of the polymerases.
00:26:13;27 I'll also point out that I haven't shown
00:26:16;03 either the sliding clamp loader, RF-C,
00:26:19;10 or sliding clamps.
00:26:20;18 They would both be acting, but, again,
00:26:22;11 they're not part of the actual replication fork.
00:26:24;20 So, I hope you've learned
00:26:27;25 a lot about how replication works, here,
00:26:30;13 and if you stay tuned I'll tell you
00:26:32;18 much more detail
00:26:35;11 about how the helicases are loading,
00:26:37;04 and what we've learned by using
00:26:38;21 single-molecule biochemistry
00:26:40;16 to investigate this process.
00:26:42;19 So, I'd like to thank Sera Thornton,
00:26:44;19 who did all the animations in this part of my presentation.
00:26:47;14 These were done as part of the development
00:26:49;28 of a course called 728x,
00:26:51;24 which is available on
00:26:54;16 the MITx and edX platforms,
00:26:56;18 and is of course focused on molecular biology,
00:26:59;10 not just DNA replication but all of the central dogma.
00:27:02;29 I'd also like to thank my funding resources,
00:27:05;02 the Howard Hughes Medical Institute,
00:27:06;28 as well as the National Institute of General Medical Sciences.

Part 3: Single-Molecule Studies of Eukaryotic DNA Replication

00:00:08;01 Hi. My name's Steve Bell,
00:00:09;24 and I'm a professor of biology at MIT
00:00:12;04 and an investigator of the Howard Hughes Medical Institute.
00:00:15;05 And what I'd like to tell you about today
00:00:17;13 is how we've used single-molecule biochemical studies
00:00:19;15 to understand a key event
00:00:21;23 in the initiation of eukaryotic DNA replication.
00:00:25;03 I first want to start, however,
00:00:27;20 by reminding you that single-molecule studies
00:00:29;05 are not new to DNA replication.
00:00:32;07 As early as the late-1960s,
00:00:34;11 Hubrin and Riggs used the incorporation
00:00:36;13 of radioactive nucleotides
00:00:38;06 to visualize individual replicating DNA molecules.
00:00:43;06 And, using this analysis,
00:00:44;20 they were not only able to identify
00:00:47;01 the fact that eukaryotic chromosomes
00:00:49;06 have multiple origins of replication,
00:00:50;25 but they also were able to demonstrate
00:00:53;11 that those origins led to
00:00:56;00 the initiation of bidirectional replication.
00:00:57;19 And ever since this key, seminal observation,
00:01:02;03 it has been an important question in the field
00:01:05;07 of DNA replication
00:01:06;20 how the bidirectional nature of DNA replication
00:01:09;26 is established,
00:01:11;06 and that'll be a theme of my presentation today.
00:01:14;07 Now, the... as I discussed in the previous part of my talk
00:01:19;18 on initiation of replication,
00:01:21;00 the initiation of eukaryotic DNA replication
00:01:23;01 is segregated into different parts of the cell cycle.
00:01:26;20 The first event, of helicase loading,
00:01:28;15 which marks all potential origins of replication,
00:01:31;19 occurs during the G1 phase of the cell cycle.
00:01:34;24 The second event, of helicase activation,
00:01:37;04 can only occur when cells
00:01:39;21 enter into the S phase of the cell cycle,
00:01:41;18 and this is triggered by the action of two kinases,
00:01:45;21 and leads to the formation of the activated helicase
00:01:47;27 and, eventually, the replisome.
00:01:49;09 And, again, if you want more details about this,
00:01:51;09 you should look at the previous presentation
00:01:53;16 on the initiation of replication.
00:01:55;20 For this presentation,
00:01:57;12 we're just going to focus on
00:01:59;16 this earliest event of helicase loading,
00:02:00;29 which I'll remind you
00:02:03;21 marks all potential origins of replication.
00:02:05;21 So, I want to give you a little bit more detail
00:02:08;03 about this event.
00:02:09;23 So, this event requires
00:02:12;11 opening of the heterohexameric
00:02:14;29 ring-shaped Mcm2-7 complex,
00:02:17;11 placing it around the double-stranded DNA,
00:02:19;22 and then closing it again
00:02:21;15 such that it encircles that double-stranded DNA.
00:02:24;18 And importantly, this occurs not once but twice,
00:02:27;15 such that you assemble an Mcm2-7 double hexamer,
00:02:31;20 with each of your N-terminal ends,
00:02:34;02 which are one end of the protein,
00:02:35;23 interacting with one another,
00:02:38;13 as is illustrated here.
00:02:39;17 Now, these are arranged in such a way
00:02:42;23 that the helicases, once activated,
00:02:45;19 will leave the origin in opposite directions,
00:02:47;18 and thus this is the first marker
00:02:49;14 of the bidirectional nature of DNA replication.
00:02:53;18 Now, the Mcm proteins don't do this alone.
00:02:55;18 They require the action of three additional proteins,
00:02:59;20 called helicase loading proteins,
00:03:02;01 called ORC for origin recognition complex,
00:03:04;00 Cdc6,
00:03:05;20 and Cdt1.
00:03:07;11 Now, a variety of studies from a number of labs
00:03:11;04 has led to an early understanding of this process,
00:03:14;13 and that's outlined on the next slide.
00:03:16;25 So, we know that the first step in this process
00:03:19;15 is the recognition of the origin DNA
00:03:21;29 by the origin recognition complex,
00:03:23;13 which binds a specific sequence
00:03:26;11 found in the yeast DNA replication origins,
00:03:28;19 and binds to other attributes of origins
00:03:31;00 that don't have specific sequences
00:03:33;26 associated with them.
00:03:35;21 When cells enter into the G1 phase of the cell cycle,
00:03:39;03 ORC is joined by Cdc6
00:03:42;02 to form a six-membered ring around the DNA,
00:03:45;12 and this can, in turn,
00:03:48;17 recruit the Mcm2-7-Cdt1 complex
00:03:51;11 to form an initial OCCM complex with the DNA.
00:03:55;06 Now, the OCCM stands for
00:03:57;19 ORC, Cdc6, Cdt1, and the Mcm2-7 complex,
00:04:00;17 so all four proteins are present at this point.
00:04:03;20 Now, at the time we started these studies,
00:04:06;21 there were a lot of questions about what happens
00:04:09;08 after the formation of the OCCM,
00:04:10;20 and this was in part due to the type of assays
00:04:13;01 that were used to monitor these events.
00:04:15;01 They typically involved
00:04:17;00 attaching magnetic beads
00:04:19;07 to many copies of origin-containing DNA
00:04:21;04 and then, to those origin-containing DNA-attached beads,
00:04:25;02 you incubate the four purified proteins
00:04:28;06 -- ORC, Cdc6, Cdt1, and Mcms --
00:04:30;22 with the DNA and then,
00:04:32;25 after various times of incubation,
00:04:34;23 you could monitor the association of the proteins
00:04:37;22 with the DNA
00:04:39;12 by simple washing away of non-associated proteins.
00:04:42;17 Now, this kind of approach worked very well
00:04:44;19 to understand the first requirement
00:04:46;21 for any of these factors.
00:04:48;03 So, for example, you could leave ORC out of the reaction
00:04:51;02 and none of the other proteins would associate,
00:04:53;05 indicating that it acted at an initial step.
00:04:55;28 In contrast, if you left Cdt1 out of the reaction,
00:04:58;17 you'd get the ORC binding,
00:05:00;07 you'd get ORC-Cdc6 interaction,
00:05:02;27 but you never got the OCCM.
00:05:05;15 The problem with this approach is that
00:05:07;19 it's very difficult to look at downstream events,
00:05:10;02 and there's two reasons for this.
00:05:11;19 One is if a protein is used twice, l
00:05:13;19 eaving it out only reveals the first step,
00:05:16;15 and the second is, if you're looking for example
00:05:18;28 at the release of a protein,
00:05:20;22 unless that happens very synchronously in the reaction,
00:05:23;02 it's very difficult to monitor that
00:05:25;24 in a, sort of... a bead-based assay.
00:05:30;10 Indeed, these sorts of multi-protein assembly events
00:05:32;16 typically do not occur in a highly synchronous nature
00:05:36;09 in a so-called ensemble reaction,
00:05:38;10 where you're adding many, many copies of the protein,
00:05:41;02 somewhere around 10^12-10^15 copies of the protein,
00:05:44;24 and a similar number of DNA molecules.
00:05:47;13 The odds that any event
00:05:49;21 is going to happen within a narrow window of time
00:05:51;11 is essentially zero.
00:05:53;01 So, we were interested in
00:05:54;29 understanding the events that occurred
00:05:57;05 after the OCCM
00:05:58;15 and in order to do this we've turned to single-molecule studies.
00:06:00;28 And the key question we wanted to ask...
00:06:03;17 key questions we were interested in answering
00:06:06;09 were, for example,
00:06:07;28 what are the fates of the proteins in the OCCM complex?
00:06:09;29 They're not part of the final double hexamer
00:06:12;22 that's formed on the DNA
00:06:14;15 that requires ATP hydrolysis to occur,
00:06:16;16 and it involves multiple steps,
00:06:18;29 but we don't know when they leave
00:06:21;07 and is there any, for example, order to their departure.
00:06:24;22 Secondly, we wondered
00:06:27;23 how many copies of these proteins
00:06:29;09 were required to load two copies of the helicase.
00:06:31;18 Was it two copies of all the proteins
00:06:33;22 or two of some, one of others,
00:06:36;19 or even possibly one of each of the proteins.
00:06:38;08 And finally, perhaps the most important question was,
00:06:41;27 how do you assemble a double hexamer
00:06:43;29 that has two copies of the helicase
00:06:46;16 in opposite orientations?
00:06:49;27 So, what's the mechanism of loading it once in one orientation
00:06:51;17 and then the second time in the opposite orientation?
00:06:55;00 So, as I said, we turned to single-molecule studies
00:06:57;16 to address this,
00:06:58;28 and I'd like take just a little period, now,
00:07:01;24 to explain the advantages of single-molecule biochemistry
00:07:03;21 and why we thought this was
00:07:06;03 a good use of our time to pursue.
00:07:09;22 So, first, before I do that,
00:07:11;29 I should acknowledge the "who" the "we" is.
00:07:13;12 So, all the studies I'm going to tell you about
00:07:15;27 were done by a very talented former graduate student,
00:07:18;06 Simina Ticau,
00:07:19;17 and they were all done in close collaboration
00:07:21;19 with Jeff Gelles' lab,
00:07:23;01 and particularly with Larry Friedman.
00:07:25;18 And not only did they build the microscope
00:07:28;16 that these experiments were done on,
00:07:29;25 but they were integral to all the analysis of the data
00:07:32;08 that Simina generated.
00:07:34;04 Okay.
00:07:35;21 So, why single-molecule studies?
00:07:37;12 First, because you're
00:07:39;23 constantly monitoring the protein associations
00:07:42;02 with individual DNA molecules,
00:07:43;18 you can see dynamic or unstable interactions
00:07:46;29 that are very difficult to see
00:07:48;28 using the bead-based assays.
00:07:50;15 Because the bead assays take 2-3 minutes
00:07:53;24 to wash away the unbound protein,
00:07:55;18 any association that lasts
00:07:58;00 less time than that
00:08:00;04 is essentially as if it never interacted at all.
00:08:02;20 Secondly, you can determine the precise number of proteins
00:08:05;13 that are acting on an individual DNA molecule.
00:08:08;10 Now, while in a bulk assay
00:08:10;07 you can get an average number,
00:08:11;14 so, for example, you could determine
00:08:13;04 if you have a picomole of DNA
00:08:14;21 and a picomole of protein that's bound to that DNA,
00:08:18;10 that on average there's one protein per DNA molecule.
00:08:21;00 But I'll point out,
00:08:22;16 if you have a less-than-efficient reaction,
00:08:24;06 you could have half the molecules
00:08:26;25 having two molecules bound
00:08:28;07 and the other half having none,
00:08:29;21 and you'd see the same average,
00:08:31;12 so you can't determine
00:08:33;06 a precise stoichiometry.
00:08:35;05 And finally, and perhaps most importantly,
00:08:37;04 the ability to
00:08:41;14 synchronize the reactions artificially
00:08:44;01 is a huge advantage of single-molecule studies.
00:08:46;01 As I mentioned earlier,
00:08:47;11 the bulk assays are intrinsically asynchronous,
00:08:50;21 but, in a way that I'm going to describe next,
00:08:53;19 you can use artificial synchronization
00:08:55;08 to create a synchronized set of molecules
00:09:00;03 and get very detailed kinetic understanding of the reaction.
00:09:03;17 So, let me just illustrate that for you.
00:09:04;26 So, in the context of a bulk assay,
00:09:07;20 you would have many DNA molecules
00:09:09;17 and you would add various proteins, here,
00:09:12;11 a blue circle and a red triangle,
00:09:13;28 and you'd allow them to interact for some period of time.
00:09:17;14 And then at the end of that time,
00:09:19;22 you would wash away the unbound proteins
00:09:21;22 and you would be left with
00:09:23;26 essentially what's illustrated here.
00:09:26;08 Now, there's two things you wouldn't know.
00:09:27;17 First of all, you wouldn't know
00:09:29;19 the history of any of these molecules,
00:09:32;00 in other words, did this blue molecule, here,
00:09:35;04 for example,
00:09:36;10 bind and then come back on again...
00:09:39;16 bind, release, and then come back on,
00:09:41;07 did it bind once?
00:09:42;22 Did this red triangle ever bind to that blue circle?
00:09:46;25 You won't know those things.
00:09:48;08 In addition, short of just leaving out a protein,
00:09:51;09 you don't know what the requirements
00:09:53;12 for two different proteins are in this process, okay?
00:09:56;04 But, most importantly, you have
00:09:58;01 no kinetic understanding of how this happened,
00:09:59;21 you just know the endpoint.
00:10:01;18 Now, if you do the same experiment
00:10:03;09 and monitor the events
00:10:04;25 on a single-molecule level,
00:10:06;15 they'll still be asynchronous, as was illustrated before...
00:10:10;04 so, this is time on the x axis
00:10:13;22 and the proteins bound, more or less, on the y axis,
00:10:16;14 and you can see, now,
00:10:18;15 we see the histories of these proteins.
00:10:19;28 We can see, at time zero,
00:10:22;06 neither DNA is bound, alright?
00:10:23;13 And then, later on,
00:10:25;20 we see a blue circle bind
00:10:27;02 and then a blue circle and the red triangle,
00:10:28;29 and then we see that the red triangle comes back off again.
00:10:31;26 Now, if we use these timeframes
00:10:34;01 that I've illustrated here,
00:10:35;14 these events would be occurring at very different times.
00:10:38;00 But we can take and artificially,
00:10:40;16 or computationally,
00:10:42;03 synchronize these events.
00:10:43;14 For example, we could say
00:10:45;15 we're going to start timing when the first blue circle
00:10:47;06 gets on the DNA,
00:10:48;18 and that's going to change everything,
00:10:51;03 because now we can look at the time
00:10:52;29 from the blue circle arriving to a red triangle binding.
00:10:56;10 And this is likely to be much more constrained
00:10:59;13 than the bulk reaction,
00:11:00;25 where we're going to have many of these events
00:11:02;15 starting at different times,
00:11:04;13 and therefore the second event occurring at different times.
00:11:06;23 And we could also, for example, ask,
00:11:09;11 how long does it take for the red triangle to fall off?
00:11:10;23 So, we start with the blue triangle...
00:11:12;04 the blue circle and the red triangle,
00:11:14;17 and then we measure the time until we just have the blue circle.
00:11:17;28 So, this gives us an ability
00:11:19;28 to get kinetic understanding of
00:11:21;25 complex biochemical reactions
00:11:23;09 that's essentially inaccessible
00:11:26;18 by conventional bulk assays.
00:11:28;07 So, how do we convert
00:11:31;12 a helicase loading assay
00:11:33;06 into a single-molecule format?
00:11:35;03 So, what we've done is to
00:11:37;05 simply exchange those magnetic beads
00:11:39;03 for a microscope slide.
00:11:40;19 And when we do this
00:11:42;20 we simply tether origin-containing DNA to the surface of the slide,
00:11:46;05 and then at the other end of the DNA
00:11:47;28 we place a blue fluorophore
00:11:49;27 that we can monitor by microscopy.
00:11:53;07 Then, we perfuse into that chamber,
00:11:55;12 that microscope chamber,
00:11:56;28 all the proteins required for helicase loading,
00:11:59;11 at least two of which...
00:12:01;10 at least one of which, sorry,
00:12:02;23 is labeled with a different colored fluorophore,
00:12:04;25 although I illustrate here that with...
00:12:07;13 uhhh, yeah, with two different colors,
00:12:09;01 a red and a green fluorophore,
00:12:10;25 green on Cdc6,
00:12:13;07 red on the Mcm proteins.
00:12:16;08 So, at this point, we use something called TIRF microscopy,
00:12:21;15 or total internal reflection microscopy,
00:12:22;28 in order to create what's called
00:12:26;04 an evanescent field of illuminated molecules,
00:12:30;14 and this evanescent field
00:12:32;05 importantly only stretches about
00:12:33;23 100-200 nanometers
00:12:35;23 above the surface of the slide,
00:12:36;29 so only molecules that are in the region
00:12:39;18 that the DNA occupies
00:12:41;01 can be monitored.
00:12:42;18 Any molecule above the evanescent field
00:12:44;27 will not be fluorescent,
00:12:48;26 because the illumination will not reach that point,
00:12:50;14 and this is very important because it prevents...
00:12:52;08 it allows the use of much high concentrations of proteins
00:12:54;19 because most the proteins
00:12:56;28 are not illuminated,
00:12:58;20 and that gets rid of lots of background.
00:13:00;11 Now, how do we monitor DNA binding?
00:13:02;27 We use a simple proxy for DNA binding,
00:13:05;04 which is the colocalization of the protein fluorophore
00:13:08;21 and the DNA fluorophore.
00:13:10;26 In this particular assay,
00:13:13;11 it indicates DNA binding,
00:13:16;17 and indeed this process,
00:13:18;19 which was originally developed between
00:13:21;17 the Gelles lab and Melissa Moore's lab,
00:13:23;13 is called colocalization single-molecule spectroscopy,
00:13:26;14 or CoSMoS.
00:13:28;16 So, how do we analyze this data?
00:13:30;12 So, this is an image of DNA molecules
00:13:34;09 that are attached to the surface of the slide,
00:13:36;13 or a field of view of those DNA molecules,
00:13:38;16 and what we do is we
00:13:40;16 identify individual DNA molecules
00:13:42;21 and then we monitor,
00:13:44;21 not the DNA fluorescence,
00:13:46;11 but the fluorescence of one of the proteins.
00:13:48;22 So, we can ask, over time,
00:13:50;22 how does the fluorescence colocalize
00:13:53;09 with each individual DNA molecule.
00:13:55;16 So, there's about 400 DNA spots here,
00:13:57;00 and you do this 400 times.
00:14:00;05 You can then create traces of the data,
00:14:03;04 in which you can see that, for example in this trace,
00:14:06;13 the Mcm proteins are, first, not associated...
00:14:09;23 this is the baseline, here...
00:14:11;13 and then you see two short associations,
00:14:13;00 and finally, over at the edge here,
00:14:17;00 a long association of Mcm proteins, okay?
00:14:19;26 And so we can do this for each of the DNA molecules
00:14:22;07 and determine a time of association for each one,
00:14:25;11 and a length of association.
00:14:28;02 Okay.
00:14:29;25 So, that's how we analyze it.
00:14:31;25 How do we know it's actually reflecting helicase loading?
00:14:33;28 So, I'm going to show you one control.
00:14:35;26 These are two movies
00:14:38;26 watching the association of the Mcms with DNA molecules.
00:14:40;28 You're not going to see the DNA molecules,
00:14:43;06 but take my word for it,
00:14:44;22 they are associating with the DNA.
00:14:46;03 And I've done one movie
00:14:48;08 in the absence of Cdc6
00:14:50;13 and the second movie, on the left,
00:14:52;08 in the presence of Cdc6.
00:14:53;20 And what I think you can clearly see is
00:14:55;20 when Cdc6 is present,
00:14:57;12 you see an accumulation of Mcm molecules
00:14:59;06 throughout the slide,
00:15:00;24 whereas, in its absence,
00:15:03;07 you basically see a few smudges of probably denatured protein
00:15:05;23 that are sort of hanging out on the surface of the slide.
00:15:08;27 Now, I've shown you this control.
00:15:10;20 I'll tell you that not only does this control work,
00:15:13;21 but if we leave out ORC or origin DNA,
00:15:15;24 or if we prevent ATP hydrolysis,
00:15:17;21 all of them inhibit this,
00:15:19;14 and these are all hallmarks, or characteristics,
00:15:22;03 of helicase loading that's been determined
00:15:24;20 in other types of biochemical assays
00:15:26;19 as well as in vivo.
00:15:29;03 So... we have an assay, how did we use it?
00:15:32;11 So, the first thing I want to address is
00:15:35;04 the stoichiometry between the origin DNA,
00:15:37;18 the helicase,
00:15:39;04 and helicase loading proteins.
00:15:40;17 So, to do this, we took advantage of the
00:15:43;05 ability to label two different proteins
00:15:45;12 and monitor the DNA at the same time,
00:15:47;06 and we focused initially on Cdc6 and Cdt1.
00:15:51;24 And the reason for that is
00:15:54;06 we knew Cdc6 and Cdt1
00:15:57;14 were essential for Mcm loading, okay?
00:15:59;05 However, in bead-based assays,
00:16:00;25 the association of these proteins
00:16:02;18 was essentially not detected
00:16:04;27 unless we inhibited ATP hydrolysis
00:16:06;28 and stopped the reaction at this OCCM step of the reaction.
00:16:11;15 Otherwise, it was as if they were never they,
00:16:13;29 and we knew that they had to be there
00:16:15;29 because they were essential for the reaction.
00:16:17;19 So, we thought the single-molecule setting
00:16:19;16 would allow us to monitor these events
00:16:21;10 and get an idea, for example,
00:16:23;05 of how many Cdc6 molecules
00:16:25;10 does it take to load two Mcm hexamers.
00:16:27;25 So, we did this, again,
00:16:30;07 by labeling either Cdt1 and Mcms,
00:16:32;21 or Cdc6 and Mcms,
00:16:34;17 with different colored fluorophores,
00:16:37;01 so we can monitor both molecules at the same time.
00:16:39;06 I'll first show you the example
00:16:41;22 with Cdt1 and Mcms labeled,
00:16:44;06 and then I'll show you an example with Cdc6.
00:16:47;24 So, I've shown you...
00:16:49;14 in this left-hand panel
00:16:51;24 you can see traces of Cdt1 association with DNA
00:16:56;01 in green, and Mcm association with DNA
00:16:58;29 in red.
00:17:00;28 And we've shifted the baseline of the Mcm trace up
00:17:03;27 so that you can see both traces simultaneously in the plot.
00:17:06;22 The first thing I hope you notice is that
00:17:10;07 the Mcms associate with the DNA
00:17:11;29 in a one-at-a-time fashion
00:17:13;28 -- we see an increase in fluorescence, here,
00:17:15;26 and then a second increase
00:17:17;26 where the second arrow illustrates.
00:17:19;18 Now, this could be because
00:17:22;11 we have two Mcms loading in the first step
00:17:25;22 and another two in the second step.
00:17:27;06 However, we can eliminate this model
00:17:29;08 and demonstrate that it's actually one Mcm
00:17:31;16 and then a second Mcm
00:17:33;08 using a process called photobleaching,
00:17:35;00 which stochastically inactivates fluorophores,
00:17:37;05 so we can take... at the end of the reaction,
00:17:40;09 we can turn up the lasers so that we see photobleaching,
00:17:42;24 and because it's a stochastic process,
00:17:45;10 if there's two fluorophores,
00:17:47;05 the fluorescence will decrease in a step-wise fashion
00:17:50;08 in two steps;
00:17:51;25 if there are four fluorophores, we'd see four steps.
00:17:53;22 And Simina has done many of these experiments
00:17:56;06 and we always see a two-step loss of fluorescence,
00:17:58;19 indicating that this is a first
00:18:01;00 and a second Mcm protein.
00:18:03;09 The other thing I think you'll clearly see is that
00:18:06;27 the time of Cdt1 association
00:18:09;03 is identical to the time of the Mcm association.
00:18:11;00 However, Cdt1 comes off rapidly,
00:18:13;12 leaving behind the Mcm molecules,
00:18:15;20 so you see a Cdt1 associated
00:18:17;26 with the first loading event
00:18:19;21 and then a Cdt1 associated with the second loading event,
00:18:22;05 but both of those are much more short-lived
00:18:25;19 than the corresponding Mcm association.
00:18:27;17 You can see that this is
00:18:30;02 indeed occurring simultaneously
00:18:31;19 in these one-second frame shots
00:18:34;08 of either the Mcm or Cdt1 fluorescence,
00:18:36;19 and you can see both of them occur within
00:18:39;17 a 1-second window of each other
00:18:41;07 getting on the DNA,
00:18:43;02 and we know this is actually true at higher time resolutions
00:18:45;15 as well.
00:18:47;14 So, that's Cdt1.
00:18:49;03 Clearly, we need two molecules.
00:18:50;20 This doesn't turn out to be surprising,
00:18:52;15 because we know Cdt1 and Mcm form a complex.
00:18:54;16 What about Cdc6?
00:18:56;15 Well, we see a very similar pattern.
00:18:58;15 You can see, again,
00:19:00;07 we have a first and a second Mcm
00:19:03;00 binding to the DNA,
00:19:05;08 and each one is associated with a Cdc6 molecule
00:19:07;11 getting on at the same time,
00:19:09;08 but coming off rapidly thereafter.
00:19:11;15 The only difference,
00:19:13;09 and this is consistent with our previous biochemical studies,
00:19:15;06 is that Cdc6 always gets on the DNA
00:19:18;22 before the Mcms get on the DNA,
00:19:20;23 usually by at least 10-15 seconds.
00:19:23;25 Okay?
00:19:25;03 So, if I summarize this,
00:19:26;24 what I would tell you is that this shows that you need...
00:19:29;19 that distinct Cdt1 and Cdc6 molecules
00:19:31;28 are associated with the loading the
00:19:34;05 first and the second Mcm complex.
00:19:36;23 Now, one thing we wondered about is
00:19:40;27 whether the release of these molecules might be ordered.
00:19:42;20 If it was ordered, it might suggest that
00:19:46;17 this is connected to the process of helicase loading,
00:19:47;28 because that would suggest that there was
00:19:50;09 some need to keep one molecule...
00:19:52;06 Cdt1, for example, after Cdc6, or vice versa.
00:19:55;12 You can see already that the synchrony of these reactions is,
00:19:59;01 in this case, not bad,
00:20:01;02 but not great,
00:20:02;29 especially if you look at the second loading.
00:20:04;17 So, we used this post-hoc synchronization
00:20:06;07 in order to synchronize the events
00:20:08;14 according to a defined event.
00:20:10;17 In this case, we chose
00:20:12;24 the arrival of Mcm on the DNA.
00:20:14;14 So, whether we're measuring Cdc6 release or the Mcm...
00:20:17;28 Cdt1 release,
00:20:20;05 we measure the time with a starting point
00:20:23;09 at t=0, so to speak,
00:20:25;27 of when the Mcm that they're associated with
00:20:28;08 bound to the DNA.
00:20:29;22 And then we asked, how long until the Cdt1 releases
00:20:32;27 versus the Cdc6 releases?
00:20:35;05 And we plotted this in the form of
00:20:37;24 a so-called survival curve,
00:20:39;20 and this just shows what percentage of molecules
00:20:41;17 are still associated with the DNA
00:20:43;20 at various time points after Mcm binding.
00:20:47;21 And I think you can clearly see that Cdc6,
00:20:50;14 on average,
00:20:52;18 releases well before Cdt1.
00:20:54;02 Now, this is done with two different experiments,
00:20:56;07 and comparing the results,
00:20:57;21 but I'll tell you that when we label Cdc6 and Cdt1
00:21:00;26 in the same reaction,
00:21:02;17 and look at their release from individual DNA molecules,
00:21:04;25 we see exactly the same result
00:21:07;04 -- Cdc6 releases before Cdt1.
00:21:10;16 So, this suggests that those release events
00:21:12;28 are actually coupled to some important element
00:21:16;06 of the reaction.
00:21:17;19 So, we also wondered,
00:21:19;26 is this the same for the first and the second Mcm molecule?
00:21:23;01 So, we did the same measurement,
00:21:24;29 and we did it...
00:21:26;28 we segregated the first Mcm loading event
00:21:28;27 and its associated release of Cdc6 and Cdt1,
00:21:31;27 and the second one.
00:21:33;27 And the clear outcome of this is that
00:21:36;15 whether you're looking at Cdc6, in blue on the left,
00:21:38;21 or Cdt1, in red on the right,
00:21:40;22 the first molecules are always released
00:21:43;13 approximately in half the time
00:21:45;23 that the second molecules are.
00:21:47;29 And this gave us the first hint that maybe the loading of the first Mcm
00:21:51;13 doesn't work in exactly the same way as the second Mcm.
00:21:54;09 Okay?
00:21:55;21 So, let me summarize what I've told you so far.
00:21:59;07 So, we know that you start
00:22:01;14 with this OCCM complex
00:22:03;15 and we end with the double hexamer,
00:22:05;08 but now we know a lot of events that occur in between.
00:22:08;01 We know that we have an ordered release of Cdc6
00:22:11;24 and then Cdt1 from the DNA,
00:22:13;08 and then we have an ordered release...
00:22:15;09 ordered assembly of a second Cdc6,
00:22:17;28 a second Mcm2-7/Cdt1 complex,
00:22:21;14 followed by another round of Cdc6
00:22:25;14 and then Cdt1 release,
00:22:27;09 albeit a little slower.
00:22:29;28 Now, you'll note, at this point,
00:22:31;19 that while we've gained a lot of understanding of Cdc6 and Cdt1,
00:22:35;08 there are two questions we haven't addressed.
00:22:37;29 One of them is the key question of
00:22:40;15 how you make a head-to-head double hexamer.
00:22:41;28 How do you load one Mcm in one orientation
00:22:44;07 and the other one in an opposite orientation?
00:22:46;02 And the second, which turns out to be related,
00:22:48;01 is how many ORC molecules
00:22:50;06 are required for this process?
00:22:51;27 Now, let me explain why they're related.
00:22:54;21 So, there are two basic models
00:22:56;20 to how you can load two Mcms in opposite orientations.
00:23:00;13 In the two model,
00:23:03;08 that would suggest that you do the same reaction twice,
00:23:06;03 with two different ORC molecules
00:23:08;00 that are bound in opposite orientations to one another.
00:23:10;19 In the first reaction,
00:23:12;13 you use the first ORC molecule to bind,
00:23:15;02 as I described before,
00:23:16;16 to the C-terminal region of the Mcm complexes,
00:23:18;08 and then hat's loaded.
00:23:21;01 And then at some point, you have a second ORC molecule bound,
00:23:23;24 and it performs the same reaction,
00:23:25;21 just in the opposite orientation.
00:23:27;10 These two Mcm complexes
00:23:29;03 then come together
00:23:30;18 and form the double hexamer.
00:23:32;21 A different model would suggest that
00:23:35;12 there's only a single ORC molecule on the DNA.
00:23:37;09 That does the first round,
00:23:39;10 as we've described before,
00:23:41;18 but that the second round is mediated
00:23:43;26 not by interacting with ORC,
00:23:46;29 but by interacting with the first Mcm protein,
00:23:49;11 using this N-terminal/N-terminal interaction
00:23:52;16 that's part of making the final double hexamer,
00:23:54;25 and I should point out that the double hexamer is very tightly held together.
00:23:58;07 So, what I think is clear is that
00:24:00;12 one key difference between these two models
00:24:02;07 is the number of ORC proteins
00:24:04;10 that would be present on the DNA
00:24:06;03 during the loading of a double hexamer.
00:24:08;08 And so Simina did the same types of experiments
00:24:10;19 I've described for Cdc6 and Cdt1,
00:24:12;29 but using an ORC molecule
00:24:17;05 and Mcm labeled,
00:24:18;25 instead of either Cdc6 and Mcm, or Cdt1 and Mcm.
00:24:21;27 And this just shows two sample traces
00:24:24;19 for those experiments.
00:24:26;08 And, again, you can see
00:24:29;04 the first and the second Mcm being loaded,
00:24:30;20 but this time we see a single ORC molecule,
00:24:33;01 a single increase in the ORC-associated fluorescence,
00:24:35;06 that lasts throughout both the first and the second
00:24:37;20 loading events.
00:24:39;10 And I can tell you, but won't show you,
00:24:41;11 that photobleaching experiments again
00:24:43;06 demonstrate that this is one ORC molecule,
00:24:45;01 not two ORC molecules binding the DNA simultaneously.
00:24:49;05 What I think you'll also notice is that
00:24:51;12 while ORC stays on the DNA after the first loading event,
00:24:53;18 it's rapidly released after the second loading event.
00:24:57;18 And this suggests that
00:25:00;22 the ORC release might be connected
00:25:02;16 to completion of a double hexamer assembly.
00:25:04;26 And we wondered whether this might be
00:25:07;11 temporally connected to some of the interactions...
00:25:09;12 release events that we looked at earlier.
00:25:11;20 So, we again determined the time of ORC release
00:25:14;16 relative to the arrival of the second Mcm...
00:25:16;28 okay, because after the first Mcm
00:25:18;27 it stays on for a very long time,
00:25:20;19 and what I think you can see is that
00:25:22;25 it's clearly distinct from the blue line,
00:25:24;20 which is the Cdc6 release events,
00:25:26;15 but it sits pretty close to on top
00:25:29;19 of the Cdt1 release events.
00:25:31;00 And in fact we can statistically show
00:25:33;13 that these are not significantly different events,
00:25:35;24 suggesting that the release of ORC
00:25:38;08 is coupled to the release of Cdt1 from the DNA.
00:25:42;05 So, this suggests that
00:25:45;15 the one-ORC model is correct
00:25:47;06 and, in order to address that more,
00:25:48;22 Simina turned to something called fluorescence resonance energy transfer.
00:25:52;13 And so what she did was to
00:25:54;20 label one population of ORC with a green or donor...
00:25:58;04 sorry, one population of Mcm proteins
00:26:00;17 with a green or donor fluorophore,
00:26:02;07 and a second population
00:26:04;08 with a red or acceptor fluorophore.
00:26:06;21 And it turns out you can use these two as a molecular ruler,
00:26:10;18 and when the double hexamer comes together,
00:26:12;17 you'll see a different level of what's called FRET
00:26:14;28 than you will when they're apart.
00:26:17;03 So, let me illustrate that here.
00:26:19;12 So, when they're apart,
00:26:21;02 when you illuminate the green fluorophore,
00:26:23;01 it will generate an emission
00:26:26;28 that has the potential to activate the red fluorophore.
00:26:30;10 However, if they're far apart,
00:26:33;20 most of that emission will not hit the red fluorophore.
00:26:36;10 In contrast, you will see
00:26:38;24 what's called FRET
00:26:40;16 when they're within approximately 10 nanometers of one another,
00:26:42;19 and then,
00:26:44;22 when the green fluorophore emits,
00:26:46;12 it will actually excite the red fluorophore,
00:26:48;27 and so even though you're exciting
00:26:51;02 only the green fluorophore,
00:26:53;05 you'll still see red emissions,
00:26:54;22 and in fact the tell-tale sign of this is
00:26:57;08 the green emissions go down
00:26:59;05 while the red emissions go up
00:27:01;07 when you're in a FRET state.
00:27:02;29 Now, importantly, when Simina set this up,
00:27:05;14 she picked sites that we knew were close together
00:27:07;09 in the double hexamer to modify,
00:27:09;21 and she could demonstrate whenever she saw FRET,
00:27:12;05 it had the hallmarks
00:27:14;20 of a loaded double hexamer.
00:27:16;10 So, the real question we wanted to address is
00:27:18;24 when does this happen?
00:27:20;21 If the model that we proposed is correct,
00:27:22;19 this should be a very early event
00:27:24;24 in forming the double hexamer.
00:27:26;17 We don't know that this particular region will be the first place
00:27:29;08 that comes together,
00:27:30;03 but it should come together relatively quickly.
00:27:33;07 And so we measured the time of FRET formation
00:27:35;06 relative to these other release events
00:27:37;18 after the second Mcm.
00:27:39;05 Recall, we can't form FRET until we have two Mcms on the DNA,
00:27:42;13 so we can't do any measurements on the first one,
00:27:44;17 but it also wouldn't be relevant.
00:27:47;05 Okay.
00:27:48;26 You can see the ORC and Cdt1 coming off
00:27:51;15 later than FRET formation...
00:27:53;01 and I should point out that this...
00:27:54;18 the FRET formation...
00:27:56;03 we have an inverted axis, so as it goes down
00:27:58;06 more and more molecules have FRET, okay?
00:27:59;29 So, you can see, in the green line on the far left,
00:28:02;20 that's the earliest event we can see.
00:28:05;15 In some cases, it's instantaneous
00:28:07;12 upon the initial association.
00:28:09;05 In other cases, it takes more than a few seconds to occur,
00:28:13;01 but it's always before Cdc6
00:28:14;19 and it's always before Cdt1 release,
00:28:16;16 consistent with the model with this being
00:28:19;03 a very early event
00:28:23;10 in the recruitment of the second Mcm molecule.
00:28:25;15 Okay, so let me summarize what I've told you so far.
00:28:28;11 Our original understanding
00:28:30;22 was this initial association event
00:28:32;23 where we had ORC, then Cdc6,
00:28:34;18 then Mcm/Cdt1,
00:28:35;29 and we formed the OCCM,
00:28:37;26 and then there was a lot of question marks,
00:28:39;16 and we only knew there was ATP hydrolysis.
00:28:41;05 Now, we have a much better understanding.
00:28:43;19 We know the first event after the OCCM
00:28:45;20 is this ordered release of Cdc6 and Cdt1,
00:28:49;01 and I'll tell you but didn't show you that
00:28:52;08 we know the ring is, in fact, closed at that point.
00:28:54;03 Then, we have a second Cdc6,
00:28:55;26 a second Mcm/Cdt1 binding,
00:28:58;11 and this leads to the formation
00:29:00;11 of the first double hexamer interactions,
00:29:03;00 and we believe that's mediated
00:29:04;25 by the N-terminal/N-terminal interactions
00:29:07;04 between Mcms,
00:29:08;29 and that's in turn followed by the release of Cdc6
00:29:10;21 and, we believe, the simultaneous release
00:29:12;28 of Cdt1 and ORC from the DNA.
00:29:15;17 And there's several properties of this model
00:29:18;18 that we like in terms of driving
00:29:20;27 the formation of a double hexamer of DNA...
00:29:23;08 a double hexamer of Mcms on the DNA.
00:29:26;12 So, one is that, because ORC stays bound
00:29:30;00 after the first Mcm is loaded
00:29:31;29 and then is only released
00:29:34;06 after the second Mcm is loaded,
00:29:36;07 you will drive the reaction towards double hexamer formation,
00:29:38;26 rather than having the possibility
00:29:41;22 of having single hexamer interactions.
00:29:43;17 And, secondly -- I hope it's obvious by now --
00:29:46;14 by using the N-terminal regions of the two Mcms
00:29:49;03 to recruit the second molecule,
00:29:50;22 you will ensure that you
00:29:52;24 always form the head-to-head double hexamer
00:29:55;09 at the origin.
00:29:56;29 Clearly, there's much more that can be done
00:29:59;04 using single-molecule studies
00:30:00;25 to understand the events of replication initiation.
00:30:04;04 For example, we can start incorporating
00:30:06;26 mutant proteins into this process
00:30:08;21 to understand when key events such as ATP hydrolysis,
00:30:12;12 or key interactions,
00:30:15;05 occur and how they influence this process,
00:30:17;20 especially kinetically.
00:30:19;18 In addition, we're very excited to use FRET
00:30:22;25 to monitor additional protein-protein interactions.
00:30:25;06 I showed you using it to monitor
00:30:27;06 the formation of the double hexamer,
00:30:28;22 but you could also use it, for example,
00:30:30;22 to monitor the opening and closing of the Mcm2-7 ring
00:30:34;07 during the loading process,
00:30:36;06 which would be one of the most key events
00:30:38;27 in understanding loading.
00:30:40;12 Finally, much of the replication process
00:30:42;29 has been reconstituted with purified proteins,
00:30:46;13 and so extending this same approach
00:30:48;11 to look at the helicase activation
00:30:50;10 and replisome assembly process
00:30:52;18 is a key goal for my laboratory.
00:30:54;24 So, I'd like to end by acknowledging our collaborators.
00:30:58;19 As I said at the start, all of this work
00:31:01;20 was done in close collaboration
00:31:03;07 with Jeff Gelles' lab,
00:31:05;13 as well as Larry Friedman,
00:31:08;00 and that they, as I said,
00:31:10;20 are integral to the analysis of all the data
00:31:13;20 as well as building the microscopes that
00:31:16;18 allow this kind of research to be done.
00:31:18;06 I'd also like to thank the Lavis lab
00:31:20;03 at the Janelia Research Campus,
00:31:22;07 who developed a number of long-lasting fluorophores,
00:31:24;15 or photostable fluorophores,
00:31:26;18 that were essential for the success of this research.
00:31:31;10 And finally, I want to thank the members of my lab.
00:31:34;12 Most of the work was done by Simina,
00:31:36;14 however, she was assisted by Nikola Ivica,
00:31:38;25 who labeled many of the proteins,
00:31:41;20 which can be one of the most challenging parts of these studies.
00:31:45;16 And finally, I just want to thank the funding sources
00:31:48;27 -- the National Institute of General Medical Sciences,
00:31:51;18 as well as the Howard Hughes Medical Institute.

Videos in this Talk
  • Part 1: Mechanisms of Chromosomal DNA Replication: The DNA Replication Fork
    Part 1: Mechanisms of Chromosomal DNA Replication: The DNA Replication Fork
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: Mechanisms of Chromosomal DNA Replication: Initiation of DNA Replication
    Part 2: Mechanisms of Chromosomal DNA Replication: Initiation of DNA Replication
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
  • Part 3: Single-Molecule Studies of Eukaryotic DNA Replication
    Part 3: Single-Molecule Studies of Eukaryotic DNA Replication
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
Speaker: Stephen Bell
Total Duration: 1:28:35
Recorded: August 2016
All Talks in Genetics and Gene Regulation

Talk Overview

Every time a cell divides, its genomic DNA must be completely, accurately and rapidly duplicated. This feat is completed by an amazing, multi-enzyme nanomachine, called the replisome. The replisome includes one DNA helicase, one RNA polymerase and three DNA polymerases, as well as numerous non-enzymatic proteins, all of which work together at the DNA replication fork. In Part 1a, Dr. Bell gives an excellent, step-by-step description of the function of each replisome protein at the bacterial replication fork.  

In Part 1b, Bell focuses on the initiation of DNA replication. At the site where replication begins, chromosomal DNA is separated into two single strands.  Two replisomes are then assembled on the DNA and they move away from each other in opposite directions.  Bell describes how the sites for the initiation of replication are identified, how the helicase is loaded and activated, and how the replisome is assembled.  As he explains, these events are significantly more complicated in eukaryotes than bacteria.

In his last talk, Dr. Bell describes an assay developed in his lab to study eukaryotic DNA replication at the single molecule level. Using this assay, Bell’s lab has determined the detailed process by which eukaryotic DNA helicase loads on DNA and begins the replication process.

Speaker Bio

Stephen Bell

Stephen Bell

Dr. Bell is Professor of Biology at the Massachusetts Institute of Technology and an Investigator of the Howard Hughes Medical Institute.  His lab studies the assembly of multi-protein complexes called replisomes that are responsible for replicating eukaryotic chromosomal DNA, and the regulation of this process to ensure that each chromosome is accurately and completely replicated… Continue Reading

Playlist: DNA Replication and Repair

Related Resources

Ticau, S., Friedman, L. J., Ivica, N. A., Gelles, J., & Bell, S. P. (2015). Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell, 161(3), 513–525.

Bell, S. P., & Labib, K. (2016). Chromosome Duplication in Saccharomyces cerevisiae. Genetics, 203(3), 1027–1067.

Costa, A., Hood, I. V., & Berger, J. M. (2013). Mechanisms for Initiating Cellular DNA Replication. Annual Review of Biochemistry, 82(1), 25–54.  

Reader Interactions

Comments

  1. Pa says

    Thank you so incredibly much for the wonderful lectures! It’s so hard to find good material on the internet ( material that I feel I can trust too! I hope this is still up to date, but realise things are constantly being discovered etc ). I love the way Professor Bell describes it all & the animations are fantastic- I like to know the ‘why’ and everything behind the mechanics, not just the basic step by step process ( I hope that makes sense!). I did molecular genetics and biotechnology at University. Unfortunately, I have been quite unwell and haven’t been able to work at all ( long term illness). I have recently had an urge to refresh and learn more about everything- so seriously thank you so much for these lectures. I have now enrolled in the Ed ex course Replication and repair that professor Bell teaches and I can’t wait to start ( mid July it commences). I really hope I am able to keep up with it after not studying for so many years. May I ask, is there something I could or should do to prepare for it ( apart from the other recommended courses)??? Thank you so much again!

    Reply

  2. Finn Kirpekar says

    Thanks!
    It is far from easy to find a “correct” representation of the replication fork – even in a simplified version. This lecture/video is outstanding in capturing details and explaining in an easily understood manner.

    Reply

  3. jl brown says

    By far the best description for my limited expertise in the area. For reference, when I was at the left coast MIT, the Okazaki story had not yet been disseminated and the DNA replication dogma , to me, was clearly flawed. I wound up going into medicine. Now I prefer genomics but there is not yet a place in genomics for me.

    Reply

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