Broken Chromosome Repair by Homologous Recombination

Transcript of Part 3: Mutations Arising during Repair of a Broken Chromosome

00:00:07.21	Hi.
00:00:08.21	My name is Jim Haber.
00:00:09.21	I'm a professor of biology at Brandeis University.
00:00:13.16	I have been talking about how cells repair broken chromosomes, and I want to talk specifically, now,
00:00:19.19	about how the repair of broken chromosomes is actually fraught with mutagenic danger.
00:00:26.25	It turns out that although repair of breaks by using homologous recombination is
00:00:34.08	far more conservative than nonhomologous end-joining
00:00:37.12	-- that would just join random segments of DNA together, and translocations that you see frequently in tumor cells --
00:00:44.00	it turns out not to be as accurate as normal DNA replication.
00:00:48.15	And understanding that has led to a great deal of understanding of exactly how repair
00:00:54.02	is taking place at all.
00:00:57.22	Okay.
00:00:59.05	So, the normal process of DNA repair that I've talked about in the previous videos is
00:01:06.12	illustrated on the left side of this screen.
00:01:09.11	A double-strand break is made; the ends of the double-strand break are resected by exonucleases;
00:01:16.13	Rad51 recombination protein is recruited;
00:01:20.15	and then a search for homology takes place, where base pairing is finally accomplished
00:01:25.15	between the invading recipient DNA and the donor.
00:01:31.20	And that base pairing facilitates, then, the recruitment of the new DNA polymerase.
00:01:37.09	And the polymerase starts to copy this template in order to patch up the double-strand break.
00:01:44.19	But this repair process is different from normal DNA replication, in that
00:01:51.00	the newly synthesized DNA is being unwound as it's being synthesized,
00:01:55.06	much in the way that RNA is unwound from DNA during transcription.
00:01:59.23	And the consequence of that is that this strand sometimes can be completely liberated.
00:02:05.16	In fact, it often is liberated so that it will pair up with the other end of the double-strand break.
00:02:11.06	That makes for an efficient repair event, where just a patch of new DNA synthesis has taken place.
00:02:17.06	But it's a dangerous point.
00:02:19.08	Because at the point where this has been liberated from its template, it's possible that
00:02:24.17	this broken segment of DNA will go somewhere else and participate in an illegitimate recombination event,
00:02:31.20	leading to some hybrid or mutagenic outcome that was not part of the original repair process.
00:02:39.28	And I'm gonna talk about events that basically hijack the system to lead to certain kinds
00:02:46.17	of errors in DNA repair.
00:02:51.27	Okay.
00:02:52.21	To learn about this in some detail, we used the mating-type switching system that
00:02:57.12	I've previously described.
00:02:59.12	This is a system in which a site-specific double-strand break made by a nuclease called HO...
00:03:03.25	analogous to the way Cas9 enzymes make double-strand breaks...
00:03:09.22	that it will cut one place in the genome, and then use a template to repair that break.
00:03:17.24	In this case, the template is a sequence called HMR, and the sequences that are normally inside HMR,
00:03:24.21	that would be used in mating-type switching, had been removed and replaced by a copy
00:03:30.17	of a Uracil-3 gene that comes from a different yeast called Kluyveromyces lactis.
00:03:36.15	And these sequences, although they're intact, are not expressed because the donor
00:03:41.03	in this situation is kept in a silent heterochromatic form,
00:03:45.20	which is the normal donor state of the HMR locus in Saccharomyces.
00:03:52.04	So, even though these sequences could be expressed, they're not expressed.
00:03:57.00	And the cells, as a consequence, are Uracil -.
00:04:00.19	If we turn on the OH endonuclease,
00:04:02.21	then these sequences are used and copied as the template sequences to go into the MAT locus.
00:04:09.01	And now these sequences are expressed, because they're Ura+, and there is...
00:04:13.04	they're no longer heterochromatic.
00:04:14.24	So, the cells can be easily screened to go from Ura- to Ura+.
00:04:18.24	They've successfully done this mating-type switch, or this analogous switching process.
00:04:25.05	But that means we could select for mutations.
00:04:28.04	And it's very easy to select for Uracil- mutations, because they become resistant to a drug called
00:04:33.28	5-fluoroorotic acid.
00:04:36.08	And so we could select for your Ura- outcomes that would be the result of some event,
00:04:42.17	switching event, that had a mutation.
00:04:45.08	So, we found a lot of these.
00:04:48.12	And a reviewer came along and said, but how do you know that these mutations didn't arise
00:04:54.08	already in the silent copy?
00:04:56.15	Maybe heterochromatic experience for the Uracil-3 gene would lead to a high level of mutation?
00:05:02.18	And we could show that wasn't the case, because if we inhibit this silencing
00:05:08.05	-- and you can do that by adding a drug called nicotinamide,
00:05:11.16	which inhibits the SIR2 histone deacetylase --
00:05:13.27	we would unsilence this locus to become Ura... expressed, and these Ura- cells became Ura+.
00:05:22.15	Because this copy is mutant, arising during the repair process, but the donor template
00:05:28.17	is still intact and is Ura+.
00:05:31.02	So all the mutations that we are looking at arose during recombination.
00:05:35.20	So, we sequenced a lot of these.
00:05:39.23	Not surprisingly, a significant fraction -- more than half of the mutations --
00:05:43.22	were base pair substitutions.
00:05:45.03	Here, they're illustrated above the DNA sequence line.
00:05:49.21	And they are single base pair changes.
00:05:53.25	The ones in black represent chain terminating, or nonsense mutations, that stop the translation,
00:06:01.00	but there are lots of base pair substitutions.
00:06:04.02	But then there were a lot of other mutations that were... that were quite different.
00:06:10.06	One very simple one is just that there are two copies of a sequence in a row, ATCATC.
00:06:15.04	And this becomes a deletion of three bases.
00:06:18.09	Somehow, the polymerases ignored or stepped over one copy of this triplet.
00:06:23.08	But there were a lot of these kinds of frameshift mutations.
00:06:29.04	About 30% of all the events were -1 frame shifts.
00:06:34.00	And that's interesting, first of all, because they all occurred in very specific contexts.
00:06:39.15	They weren't just random losses of one nucleotide.
00:06:43.07	Almost all of these mutations occurred in homonucleotide runs.
00:06:48.08	For example, here are 3 G's that become 2 G's.
00:06:52.07	And here there are 4 C's that become 3 C's.
00:06:56.02	So, what was happening frequently is that the polymerase was copying in a... in a homonucleotide run,
00:07:02.12	and just simply dropping out a base.
00:07:05.21	It's interesting also that there are no +1's.
00:07:08.08	And it turns out that this family of polymerases... and it turns out, it's DNA polymerase delta,
00:07:13.15	as I'll show you, that's doing all of this... this family of polymerases very frequently
00:07:18.06	will step over a base, making a -1 mutation, but it doesn't make +1 mutations.
00:07:26.16	But there were other, far weirder events than what I've just described.
00:07:31.07	And the two that I'm gonna focus on are the ones in the red boxes,
00:07:35.05	which are called quasipalindrome mutations,
00:07:37.16	and these long red lines that I will talk about some more.
00:07:42.09	The quasipalindrome mutations are a reflection of what I told you at the beginning,
00:07:47.18	which is that the newly synthesizing DNA is gonna fall off from its template.
00:07:52.03	And here, when it falls off from its template, it's capable of base pairing with itself.
00:07:59.02	Obvious... these are so-called quasipalindrome mutations because they're not entirely perfect,
00:08:05.06	as is illustrated here.
00:08:07.11	But if this folding over happens, and then you copy the newly synthesized strand,
00:08:12.24	you are effectively perfecting the palindrome.
00:08:15.05	And in the course, introducing complex mutations.
00:08:17.25	And we found a number of these quasipalindrome mutations, as a reflection of the fact that
00:08:23.26	the DNA polymerase that is copying the template is falling off, pairing with itself,
00:08:29.09	and then eventually getting back to doing its... finishing the job so that we could recover these events.
00:08:35.10	So, quasipalindromes are yet another reflection of the instability of this copying process.
00:08:42.20	And then there were the very weird events, where it turns out that a significant fraction
00:08:46.27	of the sequence didn't come from the original template.
00:08:50.22	And it turns out that it came from another copy of Uracil-3 on a different chromosome.
00:08:57.17	So, as I said at the beginning, we were using a copy of Uracil-3 that did not come from cerevisiae,
00:09:03.07	but comes from Kluyveromyces.
00:09:05.24	It turns out to be 72% identical to the Uracil-3 that Saccharomyces has, which is,
00:09:12.19	first of all, located on a different chromosome, and was not expressed because it is interrupted
00:09:17.13	by a transposable element.
00:09:19.06	And in a sense, we were lazy, because we didn't get rid of these sequences.
00:09:24.02	We didn't think anything would really happen.
00:09:26.16	And because we were lazy, we were a bit lucky, because otherwise we probably
00:09:30.27	never would have seen these interchromosomal template switches, which is what is going to happen
00:09:36.00	in these cases.
00:09:37.00	So, what's happening here is that, again, the newly synthesized strand is dissociating
00:09:42.02	from its template.
00:09:43.27	But here it is somehow going to a completely different chromosome, interacting with sequences
00:09:48.20	that it is only 72% identical with, copying some of that information,
00:09:53.16	and then in order to be complete, and for us to recover the events, it has to actually jump back.
00:09:58.20	So, it requires two jumps in order to be able to do this, and each one of them
00:10:04.21	involving only a 72% identical sequence between these two templates.
00:10:09.24	So, we call these interchromosomal template switches.
00:10:13.26	And they turned out to be surprisingly frequent.
00:10:18.27	Okay.
00:10:20.17	It turns out that all of these errors that I just described are the consequence
00:10:25.15	not of some special DNA polymerase... yeast cells, like mammalian cells, have a number of
00:10:31.11	so-called translesion or bypass polymerases, which they use in situations such as confronting photodimers
00:10:40.28	that are generated by UV, and in other circumstances.
00:10:44.15	But these are not the polymerases that are generating these mistakes.
00:10:48.21	The polymerase that's generating these mistakes is DNA polymerase delta itself.
00:10:54.00	And we found this out by taking advantage of a proofreading-defective mutant,
00:10:59.22	which I'll describe in just a moment as to what that does to DNA polymerase delta.
00:11:04.13	But in the absence of this proofreading activity, what we found were many more base pair substitutions.
00:11:10.22	But all those things below the line -- the -1 frame shifts, the quasipalindromes,
00:11:16.17	and the interchromosomal template switches -- are virtually gone.
00:11:19.20	So, it is the polymerase delta, the normal replicative polymerase, which is in fact
00:11:26.03	the agent of most of these mutational events.
00:11:32.00	Okay.
00:11:32.15	So, I have to say a little bit about how polymerase delta really works.
00:11:36.16	It turns out that polymerase delta actually does... has two importantly different conformations.
00:11:44.06	Every time it adds a base, it then rearranges itself to check whether the base it added
00:11:49.19	is correct.
00:11:51.00	And it will do that... and if it finds that the base is incorrect, if that base is
00:11:55.27	not properly paired, an exonuclease activity of the polymerase will then chew it away,
00:12:01.16	it'll go back to the original conformation and resynthesize the base.
00:12:05.26	So, every step in DNA replication has the potential to go back and forth between
00:12:10.22	these two conformational states.
00:12:13.03	If we delete the proofreading activity of this enzyme, it stays, we think,
00:12:19.25	locked in one of these two conformations.
00:12:21.28	And we think that that... this conformation is more processive and doesn't fall off
00:12:28.18	the DNA the way that we see in the wild type enzyme.
00:12:32.01	And as a consequence, it doesn't make frameshift mutations, and it doesn't make
00:12:36.17	quasipalindrome mutations, but it makes many, many more base pair substitutions
00:12:40.22	because it doesn't have a proofreading domain anymore.
00:12:43.21	And the beautiful experiment done by Peter Burgers and Dimitri Gordenin, which illustrates this,
00:12:48.14	is shown here.
00:12:50.06	So, here's a template.
00:12:52.20	It has a priming oligonucleotide to which the polymerase can bind.
00:12:57.12	And once the polymerase binds, it starts synthesizing DNA down the DNA.
00:13:01.24	But when it hits this blocking oligonucleotide, which is here, the replication stops.
00:13:07.05	So, we get this one very clear block in the synthesis.
00:13:12.27	The proofreading-defective mutation just keeps on going.
00:13:16.09	It goes all the way to the end of the template, which means it somehow can displace
00:13:20.22	the blocking oligonucleotide.
00:13:23.03	And I think that's a very clear indication that the reason that we don't see
00:13:29.20	quasipalindrome  mutations, or frameshifts, or interchromosomal switches is simply that
00:13:35.03	this mutated enzyme stays on its original template and doesn't dissociate in the same way that we see
00:13:42.05	in these mutagenic events.
00:13:45.07	Okay.
00:13:45.23	So, we've studied these events in a little bit more detail.
00:13:48.13	Olga Tsaponina, who was a postdoc in my lab, created a 32-base pair deletion inside
00:13:55.07	the donor locus.
00:13:56.08	So, if this template is used in a mating-type switching event, which happens most of the time,
00:14:01.02	it produces a Uracil- cell, because the 32-base pair deletion isn't corrected.
00:14:07.12	And the only way to get a Ura+ plus cell out of this arrangement is to do
00:14:11.20	this interchromosomal switch and create a chimeric protein,
00:14:15.27	which is partly Kluyveromyces and partly Saccharomyces,
00:14:19.16	where the 32-base pair mutation has been corrected by copying from this 72%-identical sequence.
00:14:30.04	One thing that Olga learned by doing these experiments was to answer a question that had...
00:14:36.18	we'd been wondering about for some time.
00:14:38.23	We wondered whether the frequent events that we were seeing, where the polymerase
00:14:44.11	fell off the DNA, if you will, was because the donor locus was held in this very highly heterochromatic state,
00:14:52.05	where it uses very highly positioned nucleosomes across this region to
00:14:57.22	prevent its transcription.
00:14:59.06	And we wondered whether polymerase banging into these highly positioned nucleosomes
00:15:03.24	was the reason that the... that we were seeing such a high level of these mutational events.
00:15:08.23	But that turns out not to be the case.
00:15:11.02	Because here, we could unsilence the locus using a mutation that prevents the silencing
00:15:16.03	and the well-positioning of these nucleosomes, and the result was that the frequency
00:15:20.20	didn't change.
00:15:21.20	So, it isn't that the polymerase is being displaced by the highly positioned nucleosomes.
00:15:27.03	It's an intrinsic instability of the polymerase itself.
00:15:32.12	Okay.
00:15:32.26	So, these sequences are only 72% identical.
00:15:36.25	And the question is, when the jumps happen, where do they go in and where do they come out?
00:15:42.16	And so we measured these questions in some detail.
00:15:47.03	The amount of DNA that's being copied turns out to be about, on average,
00:15:53.21	250 to 300 bases from the... from the second template.
00:15:57.24	But it's not... it doesn't start at the same place, and it doesn't always stop at the same place.
00:16:03.10	It's pretty well normally distributed.
00:16:05.13	And if we look more carefully at these events, as to where the strand invasion goes into
00:16:13.03	the second copy on the other chromosome, you can see that it goes into many different places.
00:16:19.14	And if you choose one of those places where it goes in to the template, then the question is,
00:16:24.21	how does it come out on the other side of the 32-base pair deletion
00:16:29.08	in order to do the correction?
00:16:30.14	And the answer is it comes out all over the place.
00:16:32.28	So, there's no preferential place where it starts, and there's no preferential place
00:16:37.05	where it ends.
00:16:38.25	And that raises a question as to what exactly is being recognized by this polymerase.
00:16:45.02	And so one thing that we... we wanted to know is, well, that's... we had been studying these
00:16:50.26	with only 72%-identical sequences.
00:16:53.28	What would happen if we made them identical?
00:16:56.13	And what we discovered is that the rate goes up 10,000-fold.
00:17:00.03	And now, 3 out of 1000 of the... of the switching events that we recover have actually done
00:17:06.22	this interchromosomal switch.
00:17:09.04	And what that means to me is that this polymerase is very frequently falling off the DNA.
00:17:15.11	Now, we have facilitated it finding a partner, because the template is 100% identical,
00:17:21.15	and then it will go back.
00:17:23.02	And again, the template is 100% identical.
00:17:25.15	And under these circumstances, we see this very high rate of these events, which I think
00:17:30.01	means that the repair polymerase is dissociating very, very frequently from its template.
00:17:40.24	So, let me just say one more thing about these microhomologies.
00:17:44.20	So, whenever it goes in or out, we could define exactly where it goes in and out by
00:17:50.23	the matches between the sequences that it was copying and the sequences where it continued.
00:17:57.06	And some of... and so these define what are called regions of microhomology between
00:18:02.14	the divergent sequences that are being used in the repair event.
00:18:06.04	And some of these sequences are being used very, very frequently.
00:18:09.28	But other sequences, which are almost as long, are being used very infrequently.
00:18:15.09	And we don't yet understand the rules as to exactly what sequences are being used to go
00:18:21.16	in or out of these sequences.
00:18:24.09	But we think it's a fundamentally important question... is to understand exactly how microhomology
00:18:30.20	is being used in this repair event.
00:18:33.13	And one of the reasons that I think it's very important to understand microhomology-mediated events
00:18:39.06	is a phenomenon in human cells called chromothripsis.
00:18:43.24	So, I... if you've seen karyotypes of tumor cells, you can... you can stain chromosomes
00:18:51.02	and see where different segments of DNA came from.
00:18:55.23	But the surprising thing is that in some cases
00:18:58.24	there's no obvious rearrangement of the chromosomal material,
00:19:02.11	because everything comes from one chromosome.
00:19:05.07	But this phenomenon, called chromothripsis, was discovered by Peter Campbell's lab
00:19:11.07	only in 2011.
00:19:12.07	It seems astonishing that something as fundamental as this could be not known.
00:19:15.18	But it could not be known until DNA sequencing was sufficiently robust to be able to
00:19:20.21	really see these kinds of events.
00:19:22.27	And what Campbell's lab first described was chromothripsis, which means chromosome shattering.
00:19:29.28	And what was happening was that one chromosome was being broken apart into dozens,
00:19:34.27	if not hundreds of pieces and rearranged, but all within the same chromosome.
00:19:39.21	And this of course could cause translocations, deletions, all sorts of crazy events
00:19:47.21	that we did not appreciate happened at all.
00:19:50.06	And the bottom part of this just illustrates this with one chromosome.
00:19:54.05	This happens to be the one highly, highly rearranged chromosome in the... in the chromosomes
00:20:00.01	of Henrietta Lacks, the woman whose HeLa cells have been used in probably 100,000 publications.
00:20:09.28	And it turns out that Lacks' chromosome 11... one copy of her chromosome 11 shows examples
00:20:16.25	of this incredible rearrangement of chromothripsis.
00:20:20.10	So, what we would like to know is, how do these events occur?
00:20:25.07	One easy way to explain these events is that they're just being joined together by taking
00:20:30.15	all the shattered pieces and putting them back together by nonhomologous end-joining.
00:20:35.04	But another possibility, which has been suggested by Phil Hastings and Jim Lupski,
00:20:42.10	and also by Gilles Fisher and Bernard Dujon,
00:20:48.11	is that there is something called microhomology-mediated template switching.
00:20:52.23	And the idea of these events is that... that it is the repair DNA polymerase which
00:20:58.21	starts to copy some region, and then jumps, sometimes a megabase away or even further,
00:21:05.10	to another place, picking up some sequence, and then going to another place and going to another place.
00:21:10.17	And stitching together a rearranged chromosome, which, again, has very small junctions,
00:21:15.24	microhomology junctions, but is a replicatively dependent process.
00:21:21.00	And we think, in fact, that the DNA repair events that we are looking at fall into this category.
00:21:27.15	I think that understanding microhomology-mediated template switching
00:21:31.03	is a fundamentally important question.
00:21:33.18	And the yeast system that I've described provides insights into what is really happening
00:21:40.00	during these template-switching events.
00:21:42.20	I've spent a lot of time talking about the individual steps in DNA repair.
00:21:47.11	We've done this mostly in the budding yeast Saccharomyces cerevisiae,
00:21:51.16	a simple unicellular organism.
00:21:53.24	But the principles of repair that we have learned about turn out to be shared by
00:21:59.09	all eukaryotic organisms.
00:22:00.26	There are differences in details
00:22:02.27	-- mammalian cells have some different proteins from those yeast has --
00:22:07.11	but the fundamental processes are really remarkably conserved.
00:22:11.13	And it becomes even more important to understand some of these steps in detail,
00:22:18.07	because all of the new gene editing techniques using site-specific nucleases
00:22:22.18	-- Cas9 in particular --
00:22:25.09	also rely, basically, on these very similar kinds of repair mechanisms.
00:22:30.17	So, understanding them, and the dangers of associated mutations associated with
00:22:36.02	these processes will turn out to be a very important thing to follow up.
00:22:41.05	Thanks very much.