Broken Chromosome Repair by Homologous Recombination

Transcript of Part 1: Broken Chromosome Repair by Homologous Recombination

00:00:07.22	Hi.
00:00:08.22	My name is Jim Haber.
00:00:09.22	I'm a professor of biology at Brandeis University, near Boston.
00:00:13.19	I'm very interested in how cells repair their broken chromosomes.
00:00:17.27	And I'm especially interested in how they use homologous recombination
00:00:21.24	to preserve genome integrity.
00:00:25.03	When we were born, we had 23 pairs of chromosomes from our mother and our father that had
00:00:31.19	a particular chromosome arrangement, and that arrangement has stayed stable through
00:00:35.26	all those rounds of mitoses in order to produce the trillions of cells that make up our body.
00:00:43.26	The exception to this genome stability is what happens in tumor cells.
00:00:48.22	And here, you can see at this microscopic level truncations, translocations, inversions...
00:00:55.04	all sorts of chromosome rearrangements.
00:00:56.24	And of course, many other alterations that you can't see without going down to the level
00:01:01.10	of DNA sequencing.
00:01:04.10	Different tumors have different rearrangements, but all of them are somehow creating
00:01:10.22	these kinds of chromosome alterations, many of which are not important in the development of the tumor.
00:01:17.00	But sometimes these rearrangements turn out to drive the cancerous nature of these cells.
00:01:22.23	One example is the so-called Philadelphia chromosome, which is found
00:01:27.24	in chronic myelogenous leukemia, where two segments of genes are joined together.
00:01:33.03	And what happens is essentially that a perfectly nice gene is turned on at the wrong time,
00:01:38.03	and drives tumor growth, when otherwise this gene would be silenced.
00:01:42.25	So, some of these translocations are actually responsible for some of the phenotypes
00:01:48.20	that cancer cells have, but many of the other rearrangements you see are just the consequence of
00:01:53.18	joining pieces of DNA together without any actual consequence for the life of the cell.
00:02:02.02	The failure of these cells to maintain their stable genotype is because they have defects
00:02:07.26	in the homologous recombination machinery that I'm gonna talk about.
00:02:11.25	As I said, they have very efficient ways of joining segments of DNA together which have
00:02:17.03	become broken, but they no longer have the ability to put these segments back together
00:02:22.08	in an orderly fashion that preserves genome integrity.
00:02:25.23	And that's really what I'm gonna talk about for the rest of this talk.
00:02:30.00	Okay.
00:02:31.03	The source of the breaks that these chromosomes have comes from replication.
00:02:37.18	It doesn't come primarily from radiation or from other external agents.
00:02:43.23	It's the process of replication itself which is... which is incredibly accurate, but, nevertheless,
00:02:50.17	every time this much DNA is replicated in the cells of your body there are breaks,
00:02:57.12	and these breaks have to be repaired.
00:02:58.27	So, an illustration of this... so, these are actually chicken cells,
00:03:02.25	but if you deprive these cells of the key recombination protein Rad51, you see all of these chromatid breaks.
00:03:11.00	And what these breaks represent is that... during the process of replication,
00:03:16.11	either the Watson or the Crick strand didn't get properly copied, and there's an interruption.
00:03:20.26	And that interruption is what is... what's seen in these... in this image.
00:03:25.16	And it's the job of the Rad51 protein, a recombination protein, to patch up this break,
00:03:31.17	which it's going to do by copying the sequences from the intact template to patch up the break.
00:03:37.28	And I'm gonna be talking in more general detail about how that process occurs.
00:03:43.18	Okay.
00:03:44.18	So, the source of most of the damage is the DNA replication machinery itself.
00:03:50.26	This is just an image of the DNA replication fork.
00:03:54.04	And really, what I'm telling you is that there are sources of damage
00:03:58.09	-- replication fork barriers and other instabilities -- that cause the replication machinery to fail,
00:04:06.26	maybe as many as a dozen times per normal cell cycle.
00:04:10.08	Maybe more, as you'll see.
00:04:12.13	So, the simplest of these breaks that arise are just when one of the two strands is nicked.
00:04:19.00	And as the replication fork comes through this sequence, it can't copy across the nick,
00:04:24.03	and this leads eventually to the formation of a double-strand break.
00:04:28.10	That essentially means that one of the two sister chromatids has a break,
00:04:32.16	and the other one is intact and could be used as a template to do its repair.
00:04:37.05	But there are other sources of these breaks that I want to just mention.
00:04:41.10	One is the consequence of UV exposure of DNA, which leads to the formation of cyclobutane dimers,
00:04:47.27	here thymine dimers, where two adjacent thymine residues become covalently linked together.
00:04:55.26	This leads to a really severe distortion of the double helix, and it prevents the normal
00:05:01.11	DNA polymerase from going through this sequence.
00:05:04.08	And that frequently, as you'll see, leads to breaks.
00:05:09.03	Another source of these breaks comes from what are called triplet repeat sequences,
00:05:14.22	here CTGCTG repeated dozens of times.
00:05:18.20	In the case of Huntington's disease, sometimes hundreds of times.
00:05:22.23	And these very simple repeats have the capability of forming quasistable secondary structures,
00:05:30.22	which, again, block the formation of the replication fork and lead to breaks.
00:05:35.26	And another example, which has become appreciated much more recently, are the formation of
00:05:41.10	what are known as RNA:DNA hybrids, or R-loops, where transcripts of DNA, which ought to be
00:05:48.09	liberated during the process of transcription so they can go off and be messenger RNA
00:05:55.00	or other kinds of RNA, remain stably base paired to the template from which they were derived.
00:06:01.16	And that... these turn out also to be severe blocks for replication fork progression.
00:06:07.11	The cell has ways of getting rid of these R-loops, either by unwinding them using
00:06:13.04	an RNA:DNA helicase, or by degrading them using some nucleases.
00:06:18.28	But quite frequently, these structures remain, and they are, again, sources of damage.
00:06:24.21	So, all of these sources of damage are at least possibly going to lead to chromosome breakage.
00:06:32.13	In humans, there have been dozens and dozens of fragile sites identified, where such breakage
00:06:40.02	is likely to occur.
00:06:42.06	One way of finding these fragile sites is by slowing down and disabling
00:06:47.20	the normal DNA replication process, in this case by using a drug called aphidicolin.
00:06:53.01	And so, if you treat the cells with a quite low dose of aphidicolin, so that replication
00:06:57.07	is proceeding, but not as efficiently as normal, you see these fragile sites appear.
00:07:03.09	Places where... again, you see sis... one of the two sister chromatids is broken
00:07:08.03	because the replication fork has been unable to get through those sequences without some kind
00:07:13.19	of consequence.
00:07:18.11	One consequence of this replication fork stalling is a phenomenon called replication fork regression,
00:07:25.03	and this will turn out to have some interesting consequences.
00:07:28.15	So, here, the replication fork is moving from left to right.
00:07:32.04	It encounters, in this case, a thymine dimer, and the replication fork can't progress
00:07:38.11	any further than that.
00:07:39.20	But interestingly, this replication fork is capable of rearrangement so that
00:07:44.14	the newly synthesized strand -- this red strand, here, and this red strand, here --
00:07:50.10	can unpair from their original template and can pair with each other to make this new structure.
00:07:55.17	And this is a very odd structure because it's got... it's a 4-way junction.
00:08:00.04	That's not the normal thing you see in DNA.
00:08:04.06	But what that leads to is the formation of these intermediate structures,
00:08:08.14	which can be seen in the electron microscope, and which are often called chicken feet for obvious reasons.
00:08:14.16	And the consequence of this is to move the replication fork backward from the place
00:08:19.28	where the stalling occurred.
00:08:21.19	One of the ways that... if this occurs, that means that maybe repair proteins can
00:08:27.16	gain access to this site and can actually repair it.
00:08:31.07	That they couldn't do if all the replication machinery was jammed up against the site
00:08:35.27	where stalling is taking place.
00:08:38.07	So, this weird structure, this chicken foot structure, has been called a Holliday Junction
00:08:44.06	after the scientist Robin Holliday who first postulated it in 1964.
00:08:49.07	As the time, nobody had ever seen these structures, but Holliday imagined what they would
00:08:54.15	look like and had very interesting predictions about how these Holliday junctions were going
00:08:59.14	to be important in recombination.
00:09:02.13	So, these structures are formed by base pairing, and they can be completely base paired.
00:09:08.24	They don't... the picture here has a little opening in the middle, but in fact every base pair
00:09:12.20	in a Holliday Junction can be paired, as is illustrated in this more accurate picture.
00:09:22.26	One of the features of Holliday junctions, which I just mentioned in a way, was the fact
00:09:27.04	that they can migrate.
00:09:28.27	And they can migrate because every time... you can form the same structure here as here.
00:09:34.24	The only difference is whether these bases are paired or those bases are paired in this structure.
00:09:41.23	And so, if you... it turns out that, energetically, it doesn't require a lot of effort for
00:09:47.19	this Holliday Junction to be able to migrate back and forth.
00:09:51.20	Here's another picture of that, illustrating the mobility of these Holliday junctions.
00:09:57.22	So that they... when they form, they can branch migrate forward and back, and they can
00:10:02.17	go away from that source of blockage and back towards that source as I mentioned.
00:10:08.17	Okay, so here again is one way in which this replication fork...
00:10:14.05	Holliday junction migration can be used.
00:10:16.20	Again, there's this chicken foot being formed.
00:10:19.23	And in this case, the 5' strand is longer than the 3' strand.
00:10:23.26	The 3' strand can now copy what was the other newly replicated strand, as shown in
00:10:29.27	the green image there.
00:10:31.04	And then, if this branch migrates all the way back, it can bypass the thymine dimers
00:10:36.15	by virtue of the fact that it has copied those sequences from a different template.
00:10:41.01	Here, the DNA damage isn't repaired, but the... but the replication fork can continue.
00:10:48.19	The other feature of Holliday junctions which is of some interest is the fact that
00:10:53.03	they can be resolved by endonucleases that will cut the Holliday junction apart.
00:10:58.20	And it can cut the Holliday junction apart in two different ways.
00:11:02.18	One way would leave the original strands in their parental configuration and be called
00:11:07.01	a noncrossover.
00:11:08.25	And the other way... which looks different, but remember that in three dimensions
00:11:14.08	these two alternative structures are in fact very similar...but if it cleaves the other strands,
00:11:20.23	then you end up with essentially a crossover, which is to say that part of
00:11:25.14	one parental strand is linked to part of the other parental strand.
00:11:28.27	And, of course, crossovers, which arise frequently in meiosis, turn out to come from
00:11:35.00	this kind of resolution of intermediate structures.
00:11:38.09	So, for the rest of the talk, I'm gonna talk a little bit more in detail about
00:11:42.24	different mechanisms of homologous recombination that can be used to patch up the double-strand break.
00:11:49.10	All of these mechanisms have one common principle, which is that the broken ends of the DNA
00:11:54.13	are going to be able to be repaired by base pairing with a template sequence, to recognize a sequence
00:12:01.01	that is identical or nearly identical, with which it can then effect repair.
00:12:07.06	The mechanism I'm gonna start talking about is break-induced replication, which is,
00:12:12.22	in a sense, the simplest of these repair mechanisms to look at.
00:12:17.19	So, I mentioned that there were these chicken feet intermediates and that a chicken foot intermediate,
00:12:23.25	in addition to being branch migrated, could also be cut.
00:12:26.22	It's a Holliday junction after all.
00:12:28.26	It can be cut by Holliday junction cleaving enzymes.
00:12:32.14	And what that does is to leave one of the of the chromatids intact and the other one
00:12:38.23	is essentially broken at the site where the replication was blocked.
00:12:43.02	So, that broken replication fork can then be used to restart DNA replication by
00:12:51.05	using a process of homologous recombination.
00:12:54.21	And the steps in this that I wanna go through are illustrated here.
00:12:59.16	So, first we have a broken replication fork, as illustrated on the top left.
00:13:05.18	The next thing that happens to this end is that enzymes -- exonucleases --
00:13:10.18	chew away one of the two strands of the DNA, leaving long 3'-ended single strands of DNA.
00:13:17.19	And those 3'-ended strands of single-stranded DNA are then bound by a recombination protein
00:13:24.22	called Rad51 in eukaryotes and RecA in bacteria.
00:13:29.16	And this recombination protein forms a filament on the single-stranded DNA, and then does
00:13:35.25	this incredible step of locating, elsewhere in the genome, homologous sequences with which
00:13:41.11	it can make those alternative Watson-Crick base pairs to recognize the template,
00:13:47.23	as illustrated on the lower left.
00:13:49.22	And this base paired intermediate, then, serves as a place where replication can be restarted.
00:13:56.00	Okay.
00:13:57.15	So, here's another illustration of this break-induced replication.
00:14:02.19	The break is made.
00:14:04.24	The end is resected.
00:14:08.05	The Rad51 protein helps to invade into the donor template, making Watson-Crick base pairs.
00:14:13.00	And then there's new DNA synthesis to restart the replication fork.
00:14:17.28	And in this case, what's illustrated is that the replication fork isn't quite normal.
00:14:23.00	Normally, we would assume that a replication fork would have leading and lagging strand
00:14:27.16	synthesis happening at the same time.
00:14:30.04	But it turns out in break-induced replication -- at least in the one case that we can
00:14:36.17	study in great detail, which is in budding yeast -- the two strands of DNA synthesis are actually discoordinated.
00:14:42.22	And this accounts for the fact that there is much more error associated with
00:14:49.02	this replication process than you would see in normal DNA replication.
00:14:52.14	In fact, in a third video that that will be part of this series, I'll talk a lot
00:14:58.02	about the errors that are produced by this kind of repair mechanism.
00:15:03.07	Okay.
00:15:04.07	So, Rad51 or RecA binds as a filament onto the DNA, and then effects this search
00:15:11.25	for homology.
00:15:15.11	If we look at these proteins in more detail, we discover that RecA and Rad51...
00:15:21.22	by each subunit of these molecules, binds three bases of the single-stranded DNA, but makes
00:15:27.22	a long and continuous filament which are illustrated here.
00:15:31.03	One of the consequences of that is to stretch the DNA by almost 50%, so that the DNA
00:15:38.15	is much more extended than it would be under normal B-DNA form.
00:15:44.18	And this stretching open of the DNA I think is very important in the way in which
00:15:48.20	this search for homology takes place.
00:15:51.08	Okay.
00:15:52.08	So, if we want to just define what's going on inside the filament, if you imagine just
00:15:57.28	cutting through the middle of the... of the filament, here's a single strand of DNA
00:16:04.16	which is being bound by the RecA or Rad51 protein.
00:16:08.17	And here it binds to double-stranded DNA.
00:16:11.12	And if it binds in the right way, then essentially all that's happening during the strand exchange process
00:16:16.28	is to exchange one base pair.
00:16:21.12	And that's happening at every step along the DNA.
00:16:24.27	But we go from having a single strand of DNA and a double strand template to having
00:16:29.27	a strand exchange intermediate and a displaced single strand.
00:16:34.01	And you can see that, also, in this biochemical example that is shown here.
00:16:39.02	So, here, in this experiment, RecA has covered a single-strand DNA template, which is homologous
00:16:46.08	to a double-stranded linear DNA, which is shown here.
00:16:50.09	And then Rad...
00:16:51.18	RecA drives the strand exchange process, forcing the pairing of one of these two strands
00:17:00.11	with the single strand, and displacing the other one.
00:17:02.20	So, at the end of this process, there's been an exchange of one strand.
00:17:06.11	The complementary strand binds to the single strand of DNA,
00:17:10.27	and the other single strand is now displaced
00:17:13.13	and is liberated as the opposite product.
00:17:19.09	A great deal of insight as to exactly how this is happening came from the
00:17:25.03	brilliant crystallographic work of Nikola Pavletich's lab, who figured out a way to crystallize
00:17:31.19	and analyze this RecA protein bound of DNA by hooking up a whole bunch of RecAs together
00:17:38.13	so that it made a uniform object for crystallographic study.
00:17:42.24	And when they did that, then they could trace the contour of the single-strand DNA inside
00:17:49.09	this RecA filament.
00:17:51.28	And what they saw was really quite remarkable.
00:17:55.28	What they saw was that the single-strand DNA was stretched.
00:17:59.21	We already knew that from electron microscopy.
00:18:03.20	But what they saw was that the stretching was not uniform, that rather than
00:18:08.13	all the bases just being pulled apart by one and a half times each three bases that were bound
00:18:16.03	by one subunit of this recombination protein are still in roughly a B-form of DNA,
00:18:23.00	and then all the stretch happens in between those three bases and the next three bases.
00:18:28.03	And this led to the understanding that the searching for homology and the mechanism
00:18:32.14	by which the strands are actually being exchanged is actually done in...
00:18:36.03	somehow, in groups of three inside each one of these subunits of the... of the recombination protein filament.
00:18:46.12	Okay, so just to summarize what this means... it's that you start with a single strand of DNA
00:18:52.11	and a double-stranded template.
00:18:55.06	And when they have lined up properly, one of those strands -- the complementary strand --
00:18:59.22	now can start to form Watson-Crick base pairs with the original single strand,
00:19:04.11	and there's the displacement of the other strand in this process.
00:19:09.17	And of course, what that means in real terms is that the... there's a formation of
00:19:14.05	what we will call a displacement or D-loop.
00:19:17.12	Here's the Watson-Crick base pairing that the Rad51 filament has made.
00:19:21.12	And this is the displaced strand, which is part of this duplex template.
00:19:27.01	And that provides the initiation for new DNA synthesis and for this repair process to take place.
00:19:34.20	So, here's the recruitment of the DNA polymerase, and then the initiation of this process.
00:19:41.00	One of the things that we learned in studying this in budding yeast, which is the place...
00:19:46.20	the only organism where these kinds of detailed molecular studies can be done so far,
00:19:53.04	is that this process requires DNA replication components
00:19:59.04	that are not essential for normal DNA replication.
00:20:01.26	So, one of these opponents is called Pol32.
00:20:06.22	It's a non-essential subunit of the DNA polymerase complex, not needed for normal DNA replication
00:20:14.05	but essential for this replication restart mechanism, break-induced replication.
00:20:19.22	And we think that this Pol32 protein is allowing DNA polymerase delta to work
00:20:25.24	as a more processive enzyme than it would under normal circumstances.
00:20:30.15	If you know the current views about DNA replication, the leading strand of normal DNA replication
00:20:38.15	is done by DNA polymerase epsilon, and Pol-delta is doing the Okazaki fragments,
00:20:43.23	which are very short.
00:20:45.15	So here, Pol-delta has to work in a much more extended way, and requires this Pol32 protein.
00:20:52.04	The second thing I can tell you about this Pol32 protein and this mechanism is that
00:20:56.28	it isn't just a yeast-specific mechanism.
00:20:59.12	It also happens in humans.
00:21:04.09	And this mechanism is called alternative lengthening of telomeres.
00:21:09.04	Many cancer cells become immortal by reactivating an enzyme called telomerase, which adds TTAGGG,
00:21:17.04	over and over, to the ends of chromosomes, their telomeres.
00:21:22.05	But some tumors don't reactivate telomerase.
00:21:25.22	And in order to keep their telomeres at a necessary length, they use recombination mechanisms
00:21:32.13	of the sort that I'm showing here.
00:21:34.10	They recombine from one telomere to another to make alternative lengthening of telomeres.
00:21:40.04	In yeast, we showed that this process required the Pol32 protein.
00:21:46.14	Much more recently, it's been shown that this break-induced telomere synthesis also requires
00:21:53.04	the homolog of Pol32, called POLD3, and is simply an illustration of the fact that
00:21:58.20	these mechanisms have been conserved all the way from Saccharomyces to humans,
00:22:05.20	you know, an enormous evolutionary distance.
00:22:07.24	Okay.
00:22:08.24	So, I...
00:22:09.26	I... that's what I wanted to say about break-induced replication.
00:22:12.20	Now, I'll say something about another process, which is called gene conversion.
00:22:17.14	And the difference here is that both ends of the double-strand break can participate
00:22:22.02	in the repair event.
00:22:23.28	And the result of this is that instead of needing to synthesize a huge long distance,
00:22:28.13	as happens in break-induced replication, just a little patch of new DNA synthesis is required
00:22:35.04	to patch up the broken chromosome.
00:22:38.20	And a mechanism by which this happens is illustrated here.
00:22:44.05	And it involves the formation of an intermediate we haven't seen so far,
00:22:48.03	which is not one Holliday junction but two.
00:22:51.05	And so, you form... by first the break, then the resection of the broken ends,
00:22:57.17	then the loading of rad51, and strand invasion... all those steps are the same.
00:23:02.10	But now, after a little bit of new DNA synthesis, which is illustrated in light blue,
00:23:07.28	you end up with a structure which has two Holliday junctions.
00:23:10.26	And this double Holliday junction can again be acted upon by resolving enzymes, by nucleases,
00:23:17.07	to end up as a non-crossover or as a crossover, and can carry out this repair process,
00:23:24.20	and just uses a little bit of new DNA synthesis, the parts illustrated in light blue.
00:23:30.09	So, these double Holliday junctions can be, again, dealt with by nucleases that can
00:23:37.17	cleave these structures.
00:23:39.10	And depending on the orientation of how these structures are cleaved, you can end up with
00:23:43.05	either crossovers or non-crossovers.
00:23:46.00	I want to just take a moment to say something about the consequences of crossovers in mitotic cells.
00:23:53.07	If these are homologous chromosomes which are undergoing repair, one of them being used
00:23:58.21	as a template to repair the other, you can end up with crossing over between these
00:24:03.28	two homologous chromosomes.
00:24:08.13	If crossover occurs between two sister chromatids, there's no genetic consequence,
00:24:13.26	because they are in fact identical pieces of DNA or... it's the identical sequence
00:24:18.12	that is just being exchanged.
00:24:20.09	But if there are crossovers between homologous chromosomes, there can be very severe consequences,
00:24:26.12	namely something called loss of heterozygosity, which is illustrated here.
00:24:31.19	So, here I'm illustrating what happens in the... when one of these two chromosomes
00:24:37.12	carries a recessive mutation called rb.
00:24:42.06	Cells that are heterozygous for this mutation don't have a phenotype.
00:24:46.10	But in cells where there has been a crossover between the two homologous chromosomes,
00:24:52.13	this makes... this causes the possibility, after chromosome segregation,
00:24:57.16	of ending up with a chromosome which is homozygous for this rb mutation.
00:25:02.20	And this loss of heterozygosity is associated with the progression of this particular disease,
00:25:08.18	which is called retinoblastoma.
00:25:11.15	But this same principle applies to a large number of other human diseases.
00:25:19.14	It turns out that a number of diseases -- retinoblastoma;
00:25:24.14	the deficiencies in breast cancer... BRCA1 or BRCA2, the two familially inherited breast cancer mutations;
00:25:31.28	something called Lynch syndrome -- are all intrinsically recessive mutations.
00:25:38.09	They don't... they don't have a real phenotype when there's a wild type copy around.
00:25:44.22	But the fact is that these kinds of recombination events occur frequently enough that some cells
00:25:51.14	in a tissue can become homozygous, have a loss of heterozygosity, so that you end up...
00:25:58.14	the people who carry these mutations, even though they're just heterozygous to start with,
00:26:02.20	end up with tissue which becomes homozygous, has a loss of heterozygosity, and that is...
00:26:08.18	it is in those tissues that these diseases become manifest.
00:26:15.06	We can also actually look at recombination visibly, by using studies of sister chromatid exchange.
00:26:25.06	Here, some fraction of the thymidines of the DNA are replaced by an analog called bromodeoxyuridine.
00:26:34.07	And so, if you start with cells that have grown in the presence of bromodeoxyuridine
00:26:38.18	for a long time, and then you take away the bromodeoxyuridine, after one round of
00:26:45.03	DNA replication this is just the picture that Meselson and Stahl showed for normal replication
00:26:50.13	in E. coli, namely that one of the strands is old and has bromodeoxyuridine,
00:26:55.17	and the other strand is new and doesn't have any bromodeoxyuridine.
00:26:59.15	And then, if these cells go through yet another round of replication, only one of the four strands,
00:27:04.11	and therefore only one of the two sister chromatids, has any bromodeoxyuridine label.
00:27:10.19	But if there's been an exchange event, a crossover, during the process of this...
00:27:18.13	of this second round, now there will be some bromodeoxyuridine on one chromatid,
00:27:24.15	but at the other end they'll be bromodeoxyuridine on the other chromatid.
00:27:28.14	And you can actually see this by staining these chromosomes for the presence of bromodeoxyuridine.
00:27:34.28	If there's no sister chromatid exchange, then you see a single continuous line of labeling
00:27:40.13	on one of the two sister chromatids.
00:27:42.28	But if there's been a sister chromatid exchange, now some of that label is exchanged
00:27:47.12	to the other sister, as you can see here.
00:27:49.14	And this turns out to be a very potent way of understanding how often these events
00:27:55.19	happen in cells.
00:27:58.06	And it turns out they're frequent, surprisingly frequent.
00:28:03.22	You can see in virtually every replication cycle in human cells that there are
00:28:09.05	a few of these sister chromatid exchange events.
00:28:12.21	If you treat these cells with a DNA-damaging agent, so that they suffer lots of chromosome breaks
00:28:19.19	that require recombination, you actually can produce these astonishing pictures
00:28:24.11	of what are called harlequin chromosomes.
00:28:26.23	And they're called harlequin chromosomes because a figure from the Renaissance,
00:28:33.07	a Commedia dell'Arte figure known as Harlequin, wore a costume of these, as shown here,
00:28:39.12	that resembles this picture.
00:28:42.02	So, this tells you that there can be many, many sister chromatid exchange events.
00:28:48.04	Normally, cells can handle this, and therefore you only see a few of these exchange events
00:28:56.14	under normal circumstances.
00:28:58.03	Okay.
00:28:59.11	And one of the reasons you only see a few of these events is that these intermediates
00:29:03.25	have an alternative way of being resolved that I haven't talked about until now.
00:29:08.17	And that is that some of these double Holliday junctions can be dissolved rather than resolved.
00:29:15.11	That is to say, they are not being cut by nucleases in the crossover and non-crossover outcomes.
00:29:21.06	They're actually being unwound and taken apart in such a way that they result in no crossing over.
00:29:27.28	And it turns out that a key element in that unwinding process is a helicase, a DNA unwinding protein,
00:29:35.23	called the Bloom helicase, which was identified by the fact that individuals
00:29:41.02	lacking this helicase are cancer-prone and have many other problems,
00:29:45.13	the so-called Bloom syndrome... which unwinds the structure so that there are no crossovers.
00:29:54.10	And so in the absence of the Bloom helicase, there's a huge increase in sister chromatid exchange.
00:30:00.28	Because nothing is being unwound, everything is being driven through the crossover pathway.
00:30:05.26	And so if you do the same bromodeoxyuridine label that I showed before, now,
00:30:11.06	when you look at the Bloom chromosomes, they're harlequin chromosomes.
00:30:13.21	They have dozens and dozens of these crossovers through their genome.
00:30:19.11	And this tells you, actually, that there are lots of breaks in DNA during normal DNA replication,
00:30:24.27	but almost all of those breaks are handled in a way that has no genetic consequence whatsoever.
00:30:29.16	Okay.
00:30:30.16	I'll just add here that one of the things we don't understand
00:30:34.06	-- one of many things we don't understand in detail --
00:30:37.21	is why these double Holliday junctions are mostly always
00:30:40.24	resolved as crossovers rather than non-crossovers.
00:30:43.24	This is something that people really are working hard to really understand.
00:30:48.18	Okay.
00:30:49.26	And then, just to complicate your life, this is not the only double-strand break repair mechanism
00:30:58.08	where both ends can participate in the repair event.
00:31:01.24	There's yet another process called synthesis-dependent strand annealing.
00:31:05.17	Again, the two ends are attacked by nucleases, become single-stranded DNA.
00:31:11.07	Rad51 protein gets involved and drives the formation of these displacement loops that...
00:31:17.03	by Watson-Crick base pairing.
00:31:19.15	There's the initiation of new DNA synthesis, as illustrated in the light blue.
00:31:23.20	But here the new DNA synthesis that's being generated is different from what happens
00:31:30.03	in the other mechanism, because it's being unwound from the template in the same way that RNA
00:31:35.12	would normally be unwound from the... from its DNA template.
00:31:39.06	And this unwound strand of DNA, the newly synthesized DNA, eventually is copied
00:31:44.23	far enough so that can the anneal with its partner.
00:31:48.23	And then it all gets patched up.
00:31:50.24	The result of this is there never was a stable Holliday junction intermediate,
00:31:55.08	and all of these events are resolved as non-crossovers.
00:31:58.21	This mechanism turns out to be very important in mitotic cells.
00:32:03.07	This mechanism, the second mechanism, turns out to be much more important in meiotic cells.
00:32:10.17	In meiosis, crossovers are of course desirable to generate genetic diversity.
00:32:16.17	But it turns out also that crossovers are necessary to hold these pairs of
00:32:23.04	homologous chromosomes together for proper chromosome segregation.
00:32:26.20	If there's no crossing over, there is what is known as first division nondisjunction.
00:32:32.06	If you look at Down syndrome individuals, who have an extra copy of chromosome 21,
00:32:39.18	they arise on chromosomes that have not had proper levels of chromosome exchange.
00:32:45.14	And so crossing over not only fulfills a diversification role, but it also turns out to be critical
00:32:53.18	in terms of proper chromosome segregation.
00:32:57.10	So, in meiosis, this unwinding pathway that I've talked about is disabled.
00:33:04.04	There are mitotic-specific proteins that basically prevent the unwinding process from happening.
00:33:11.01	That drives them into crossovers.
00:33:13.20	And so, almost all the crossovers are generated by this double Holliday junction mechanism.
00:33:19.15	And the non-crossovers turn out to be generated, for the most part,
00:33:23.19	by a synthesis-dependent strand annealing mechanism.
00:33:27.22	Okay.
00:33:29.04	Just to finish up, I'll say that there's one more interesting homologous recombination process,
00:33:34.02	and that's called single-strand annealing.
00:33:36.05	It's really the simplest of all of these events, because it just involves the break
00:33:41.06	and the resection of the break by nucleases until flanking homologous sequences are exposed,
00:33:49.10	Watson on one strand, Crick on the other.
00:33:51.12	And these can anneal to form a structure that is then trimmed.
00:33:55.21	And the result of this is a deletion between two flanking repeated sequences.
00:34:00.23	Sometimes these sequences can be dozens of kilobases or more apart, so you can make
00:34:05.22	quite large deletions between these kind of flanking repeated sequences.
00:34:11.16	These events don't need Rad51, but they do need an annealing protein called Rad52.
00:34:17.27	And the reason that these are important in people is that our genomes are littered
00:34:22.07	with repeated sequences.
00:34:23.20	There are 500,000 copies of a sequence called a Alu, 300 base pair chunks of DNA
00:34:30.26	littered around the chromosome.
00:34:32.16	And if you make a break in between them, they make deletions by single-strand annealing.
00:34:37.21	And it turns out that many human diseases have the pattern of being... of these recurrent deletions,
00:34:45.03	which are occurring between these flanking repeated Alu sequences,
00:34:50.20	and turn out to be clinically very important.
00:34:52.24	Okay.
00:34:53.24	So, I've told you about several mechanisms of homologous recombination,
00:35:00.14	which all play a role in maintaining the stability of the genome.
00:35:05.06	When these are disabled, you end up with rearrangements
00:35:09.18	which are driven by non-homologous recombination, which I haven't talked in detail about at all.
00:35:16.05	But these homologous recombination mechanisms are really the gatekeepers to the maintenance
00:35:21.09	of genome stability.
00:35:23.15	In the next video, what I will talk about is looking at these processes in more detail,
00:35:28.27	at the molecular level.
00:35:30.09	How do we know, in molecular detail, what I just told you in general terms?
00:35:35.19	And I urge you to tune in and see what I have to say.
00:35:39.24	Thanks a lot.