Broken Chromosome Repair by Homologous Recombination

Transcript of Part 2: Molecular Mechanisms of Repairing a Broken Chromosome

00:00:07.23	Hi.
00:00:08.23	My name is Jim Haber.
00:00:09.23	I'm a professor of biology at Brandeis University, near Boston.
00:00:13.11	I'm talking today about how cells repair their broken chromosomes.
00:00:18.28	I previously presented a video about the overall mechanisms
00:00:23.07	by which this double-strand break repair takes place.
00:00:27.03	And what I want to do now is to talk about how we know many of those steps in molecular detail.
00:00:36.24	Just to remind you, I talked before about the fact that tumor cells are really distinct
00:00:43.08	from the rest of the cells of our body because they have failed to maintain the proper arrangement
00:00:48.24	of the 23 pairs of chromosomes that we have in our body.
00:00:53.02	And these tumor cells suffer hundreds of chromosome rearrangements.
00:00:59.08	And these rearrangements are done by joining, inappropriately, segments of DNA from
00:01:04.22	different chromosome breaks together to make these translocations.
00:01:09.00	And there's... as a consequence, what this means is that these tumor cells,
00:01:13.15	though they are efficient in repairing DNA in one way, they've lost the accurate
00:01:18.06	homologous recombination mechanisms by which double-strand breaks can be repaired.
00:01:22.20	And that's the topic that I'm pursuing.
00:01:25.16	And as I also mentioned previously, the source of most of the double-strand breaks that lead
00:01:32.19	to these chromosome rearrangements are not from exogenous sources such as X-rays.
00:01:39.06	They come spontaneously from the process of DNA replication itself.
00:01:44.02	And I illustrated that by the fact that if you deprive these cells of their key recombination
00:01:50.13	protein, called Rad51, within a single cell division what you see are all of these chromatid breaks.
00:01:57.14	And these chromatid breaks therefore represent cases where one sister chromatid is
00:02:02.19	completely properly replicated, but its sister has an interruption.
00:02:05.26	And it's the job of the Rad51 protein to patch that up.
00:02:10.06	And I'm going to talk, now, in more detail about one way in which this occurs.
00:02:16.25	And I'm gonna talk about it using a model organism, Saccharomyces cerevisiae,
00:02:22.17	where we have the highest level of resolution of understanding this repair process.
00:02:28.17	Okay.
00:02:29.17	I also mentioned that there are multiple different pathways by which double-strand breaks
00:02:35.07	can be repaired.
00:02:36.21	There is a so-called double Holliday junction mechanism, which can lead either to crossovers
00:02:42.02	or non-crossovers.
00:02:43.27	And there is a synthesis-dependent strand annealing mechanism, which only leads to non-crossovers.
00:02:50.23	I'm gonna be predominantly talking now about this mechanism, and the way we know
00:02:57.20	how each of these steps takes place.
00:03:00.01	Okay.
00:03:01.01	So, the model system that my lab has studied for quite some time is
00:03:07.11	this so-called mating-type switching system within Saccharomyces cerevisiae.
00:03:13.07	Saccharomyces... budding yeast has two mating types: one called a and one called alpha.
00:03:19.25	And they differ by a set of about 650 or 700 bases of unique information,
00:03:27.00	so that MATa has a set of sequences that MATalpha does not have,
00:03:33.01	and they specify whether the cell behaves sexually as an a or as an alpha.
00:03:39.08	But it turns out that yeast are homothallic.
00:03:44.11	This is a term that applies to quite a number of different organisms.
00:03:49.06	They are able to change from one mating type to the other.
00:03:52.19	And this process of mating-type switching turned out to be an opportunity for
00:03:57.24	learning about how double-strand break repair takes place in detail.
00:04:02.21	In the case of this mating-type switching, there is a site-specific endonuclease,
00:04:07.17	an enzyme encoded by a gene called a HO -- homothallism --
00:04:12.20	that cuts the DNA just next to those red sequences.
00:04:17.06	And it leads to the replacement of the red sequences by a copy of blue sequences.
00:04:22.10	And obviously, for that to happen, there had to be a source for those additional DNA sequences.
00:04:28.24	And it turns out that there are two other copies of mating-type information.
00:04:33.09	Here, one called HMLalpha.
00:04:35.08	And here, one called HMRa.
00:04:38.20	And these serve as a library, or reservoir, of sequences that can be used to replace
00:04:45.05	the red sequences by the blue sequences,
00:04:47.24	and it turns out, also, to replace the blue sequences back to the red sequence.
00:04:51.11	So, the organism can switch in both directions.
00:04:55.23	What makes these donor sequences so unusual is that they are
00:04:59.24	unexpressed copies of mating-type information,
00:05:03.20	and they are kept in a highly heterochromatic and silent state by
00:05:08.28	very highly positioned nucleus zones that go across this region,
00:05:12.23	which requires a special histone deacetylase called SIR2.
00:05:17.17	So, these sequences are not transcribed, but they can be used as donors in this repair event.
00:05:26.14	And all of the expression of the mating-type information comes from the MAT locus itself.
00:05:31.28	Okay.
00:05:33.18	So, Ira Herskowitz's lab made this all possible by making a galactose-inducible version of
00:05:43.14	the HO endonuclease.
00:05:44.20	So, this enzyme, now, is only turned on when we add a particular sugar to the medium.
00:05:50.15	And when you add the sugar to the medium, very rapidly this sequence is cleaved by the enzyme.
00:05:57.19	And you can see that by using a Southern blot, which is shown here, where the...
00:06:03.03	originally, the MATa locus is cut into a smaller fragment, which you can see here.
00:06:08.17	And then, after some time, what we discovered is we could follow the appearance of the product,
00:06:15.06	because the blue sequences have different restriction sites than the red sequences.
00:06:19.17	And so, on a Southern blot, we were able to sort of follow, in real-time,
00:06:24.08	how this switching process took place.
00:06:26.26	So, the key element of this is that the endonuclease is incredibly efficient.
00:06:31.20	Almost all the sites are cleaved in a very short period of time, 20 minutes.
00:06:35.21	And after that there's a rather lengthy period of time before we see the appearance of the product.
00:06:43.05	And that allowed us to identify lots of slow steps in this process, each one of which represents
00:06:49.12	a key intermediate step in the process of DNA repair.
00:06:54.04	Okay.
00:06:55.04	The first of these that we identified was the fact that the ends of the double-strand break
00:07:01.00	are chewed away by exonucleases.
00:07:04.16	And Charles White, who was a postdoc in my lab, showed that he could see
00:07:11.09	if he separated the DNA on a denaturing gel, so that the two strands -- Watson and Crick strands --
00:07:15.22	are separate from each other, he could see the appearance of these strange,
00:07:20.13	what looked like partial DNA digests in the... in this Southern blot.
00:07:26.12	And they came because the enzyme, StyI, that he was using cannot cut single-stranded DNA.
00:07:33.07	So, it could cut the sequence at the beginning, but once the DNA was being resected
00:07:40.07	by these exonucleases, now this site became unavailable for cleavage.
00:07:44.14	And that means that we could see a DNA fragment that started at the next StyI site,
00:07:50.03	or even the next StyI site, and he could identify the fact that resection was taking place.
00:07:55.27	And this was the first real demonstration that the resection was happening in a polar fashion.
00:08:01.09	It turns out that, as far as we can tell, all resection goes in this 5' to 3' way.
00:08:08.14	It turns out there are at least three major DNA resection machines, if you will.
00:08:16.25	One of them involves a set of proteins called Mre11 and Rad50, and another protein
00:08:23.02	called Sae2 or CtIP.
00:08:25.26	It does very short resection, and it then allows other resection machineries to be
00:08:31.18	more efficiently loaded onto the broken DNA, one of which is an enzyme called Exo1,
00:08:37.06	which chews off individual nucleotides as it goes down the DNA.
00:08:41.18	And the second is a complicated quartet of proteins, which uses a helicase called Sgs1,
00:08:50.27	which is the homolog of the Bloom's helicase in people.
00:08:54.18	It's prying open the DNA, and then an enzyme called Dna2 is clipping off the DNA so there
00:09:00.20	is what is called a helicase and an endonuclease, which is cleaving off chunks of DNA at a time.
00:09:06.19	But by one of these means, then, we end up with single-stranded DNA that the Rad51 protein
00:09:12.01	is going to use to carry out the next steps in recombination.
00:09:16.04	Okay.
00:09:17.15	Here's just another illustration of this kind of resection.
00:09:23.28	We realized, and other people realized, that you could see this resection much more dramatically
00:09:28.20	if you prevented the recombination from taking place.
00:09:31.25	And so either by deleting the HML and HMR sequences, so there are no donors,
00:09:37.23	or in this case by knocking out the Rad51 protein itself, so there is no recombination.
00:09:42.19	Now you could see these bands appear on the... on the gel because resection is just inexorably
00:09:49.03	going down the DNA, making one site after another not able to be cut by the...
00:09:55.17	by this restriction enzyme, and leading to these apparently large fragments of DNA.
00:10:01.02	So, that allowed us to sort of look at this process and to figure out that, in general,
00:10:06.06	the nuclease is moving about one nucleotide a second, four kilobases an hour.
00:10:11.23	So, it's a pretty slow enzyme compared to many biochemical reactions.
00:10:19.27	Okay.
00:10:22.12	Another thing that is shown here is that if you get rid of the two machines
00:10:26.26	-- the Sgs1-Dna2 machine and the Exo1 machine --
00:10:30.11	now basically all that resection stops, except for a little bit,
00:10:33.13	which is being done by the... by the last enzyme in this set, the Mre11 complex.
00:10:44.10	In order for this to take place in chromatin... because, after all, we're not really interested
00:10:50.23	in what happens in naked DNA, but what's happening in chromatin in the nucleus...
00:10:57.04	it turns out that there have to be other components.
00:10:59.19	Because these nucleases have a very hard time getting past highly positioned nucleosomes.
00:11:06.06	And so along comes another enzyme, which is known as FUN30.
00:11:12.12	Its name came from its original identification as "Function Unknown Now",
00:11:18.21	and the name hasn't been changed.
00:11:21.09	But what FUN30 turns out to be is a chromatin remodeler, a member of the family of Swi2/Snf2 homologs,
00:11:29.15	which can open and push nucleosomes around so that it is possible for these nucleases
00:11:36.25	to get past these nucleo... these barriers.
00:11:42.04	And so, we've looked at that kind of a process in another way of measuring resection.
00:11:47.18	And that is to use small PCR reactions to say when... to say, what is the state of
00:11:57.09	the DNA at this location versus what's the state of DNA, for example, here?
00:12:02.18	And of course, what happens is that a PCR will copy any template, but in this region
00:12:08.17	here it's all... there are two copies of the template.
00:12:12.06	And in this region here, there's only one copy of the template.
00:12:15.11	So, the intensity of the PCR reaction is going to go down.
00:12:19.04	And you can see over time that the amount of resection is just getting broader and broader
00:12:24.14	as the nucleases keep chewing away the DNA at this rate of about 4 kilobases an hour.
00:12:32.13	If you do this same reaction in the fun30 enzyme deficient cells, which is shown here,
00:12:39.12	now you can see that the resection is highly confined, because it can't just go past
00:12:44.00	all of those other nucleosomes.
00:12:45.27	And there's about a four-fold reduction in the rate of resection.
00:12:50.04	So, what I've talked about so far is the fact that there's a break; the break is made
00:12:55.12	by a site-specific nuclease; the nuclease-induced break is then chewed away by a series of exonuclease
00:13:02.09	enzymes to make single-stranded DNA.
00:13:04.14	Now, I'm gonna talk about the next step in the process, which is how the Rad51 recombination protein
00:13:10.04	gets loaded onto the DNA, and then how it carries out this really critical part
00:13:15.16	of the repair process, which is this strand invasion step, where this single strand of DNA
00:13:20.06	finds a homologous template elsewhere in the genome, with which it can make
00:13:25.08	the base pairs as the initial step in the DNA repair process.
00:13:29.21	Okay.
00:13:30.21	So, I mentioned before that the Rad51 protein and the... and its E. coli RecA homolog
00:13:40.28	were understood by the really brilliant X-ray crystallography of Nikola Pavletich's lab, in order to be able
00:13:47.13	to see how the single-stranded DNA is bound inside this filament of Rad51 proteins.
00:13:55.01	And then, inside this filament of Rad51 proteins, it effects an exchange of base pairings
00:14:02.08	such that what was originally a single strand of DNA now is duplex, and it has displaced
00:14:07.20	from the original duplex DNA a single strand of DNA.
00:14:14.03	To be able to really see how Rad51 binds to broken DNA in vivo, we needed another technique.
00:14:22.13	This is too small to see microscopically.
00:14:25.21	Even with fluorescent proteins, you can't see enough detail.
00:14:30.16	And so we turned to a process called chromatin immunoprecipitation, which I'll briefly describe here.
00:14:38.23	It turns out that if you treat cells with formaldehyde, which is a cross-linking reagent.
00:14:44.12	It will join proteins to proteins.
00:14:47.19	It will also join proteins to DNA.
00:14:50.17	And they are held in this covalent state.
00:14:52.24	If the DNA protein complexes that have been cross-linked in this way are fragmented,
00:14:59.05	either by sonication or some other means, now what you end up with are relatively small pieces of DNA
00:15:05.19	to which proteins are cross-linked.
00:15:08.07	And if you have an antibody against a particular protein -- in this case, Rad51 --
00:15:14.21	you can immunoprecipitate only those segments of DNA to which Rad51 is cross-linked.
00:15:20.22	And so you pull down and purify a small subset of all the DNA to which Rad51 is cross-linked.
00:15:29.00	Then magically, by just raising the temperature to 60 degrees and holding the cells...
00:15:34.02	the solution for a while, these cross-links reverse.
00:15:37.06	And as a consequence, you can then get rid of the protein and purify the DNA
00:15:41.26	in such a way that you can ask... either by PCR or by sequencing, you can ask what pieces of DNA
00:15:48.04	have been preferentially recovered by chromatin immunoprecipitation.
00:15:54.05	Okay.
00:15:54.19	So, we did that to look at, when does the Rad51 protein become bound to the single strand of DNA
00:16:01.20	which is generated by resection, as illustrated here?
00:16:06.06	And so, here's a Southern blot.
00:16:08.20	It's spread out at the beginning to show very early times, and to show you how efficiently
00:16:13.05	the HO endonuclease is cleaving the DNA into this smaller fragment.
00:16:18.09	And then on the top is PCR directed against the sequences adjacent to the mating-type locus
00:16:24.13	to ask, when did we... when... at what point could we immunoprecipitate Rad51-bound DNA
00:16:31.23	and then analyze it?
00:16:35.00	And you can see here that we could get a robust signal about a half an hour after we started
00:16:41.00	the process.
00:16:43.18	So, that's interesting, because we can see the cut at twenty minutes, and we don't see
00:16:51.26	the Rad51 binding for another ten minutes.
00:16:55.10	There's a... there's another series of slow steps that happens before the Rad51 is
00:17:00.10	firmly bound to the single-stranded DNA.
00:17:04.25	What that other step is it turns out -- and I’m not going to show you the data for this --
00:17:08.13	is that before Rad51 binds another protein, a single-strand DNA-binding protein
00:17:14.28	called RPA first binds to the single-stranded DNA, and then it's replaced by Rad51 in a process
00:17:21.09	that requires some additional proteins, one of which is called Rad52, and in humans is called BRCA2.
00:17:28.19	And I'll show you a little bit more about that in a minute.
00:17:33.07	Okay.
00:17:34.07	So, here is the mammalian version of this kind of experiment, but now without
00:17:41.07	being able to see a specific DNA sequence.
00:17:44.19	This is just irradiating cells, and so every cell has chromosome breaks after X-ray irradiation
00:17:50.24	or... but the breaks are random with respect to any given DNA sequence.
00:17:56.09	And the advantage of the yeast system was we were looking at a specific site,
00:18:00.12	and could really define it in some detail.
00:18:02.16	But you can see here that if you... you can see the appearance of fluorescent foci,
00:18:09.10	which is by done by looking at an antibody against the Rad51 protein.
00:18:14.04	And so when you treat the cells by irradiation, you can see the formation of these foci.
00:18:19.24	Those foci don't happen very efficiently when you prevent the resection of the DNA ends.
00:18:26.17	They require single-stranded DNA for Rad51 to bind.
00:18:30.03	So, if you block the expression of a protein called CtIP, which is one of those enzymes
00:18:39.05	that is necessary for resection to take place, now you don't see these foci.
00:18:45.22	And as I said, Rad51 requires help in order to load itself onto the DNA.
00:18:51.22	In mammals, the key protein that is necessary for this is the BRCA2 protein.
00:18:59.02	This is the protein that's often mutated in women who have a predisposition to breast cancers,
00:19:05.21	a familial predisposition to breast cancers.
00:19:10.17	And you can see again that in the... in the irradiated cells you can see all these Rad51 foci,
00:19:16.11	but in the absence of the fully functional BRCA2 protein, now these foci don't appear.
00:19:24.08	And the same turns out to be true for another protein, which is a so-called paralog of Rad51
00:19:31.03	called XRCC2.
00:19:32.03	So, you need these additional proteins in order to get Rad51 to load, just as you do
00:19:37.25	in yeast.
00:19:38.25	So, in yeast, we did this by chromatin immunoprecipitation, as I showed you before.
00:19:44.14	And it turns out that in the absence of the Rad52 protein, you don't get this loading,
00:19:50.15	because Rad52 is a necessary part of the loading process.
00:19:54.12	On the other hand, another radiation-sensitive mutant in yeast, called Rad54,
00:20:00.16	turns out not to be required for this.
00:20:02.28	In the absence of Rad54, we have Rad51 loading.
00:20:10.28	This cross-linking technique, this chromatin immunoprecipitation technique, turned out
00:20:15.02	to be also... very... incredibly valuable for looking at the next step of this process.
00:20:21.08	So, after Rad51 has bound to single-stranded DNA, the next step in the process is to
00:20:28.24	search the genome to find a partner sequence with which it can make these base pairs.
00:20:34.13	And of course, in the mating-switching system, that would mean that the broken DNA at MAT
00:20:38.21	is going to find either HML or HMR, those two donor sequences,
00:20:42.18	in order to do this base pair exchange and to set up the rest of the repair, to switch one...
00:20:49.14	from one mating type to the other.
00:20:51.02	And it turns out that since you can cross-link protein to DNA, not only could we see
00:20:58.17	the cross-linking of the Rad51 protein to the original break, but once the strand invasion
00:21:04.10	had taken place, now we could see that the cross-linked proteins would not only immunoprecipitate MAT,
00:21:11.03	but they would immunoprecipitate the HML locus, the donor locus as well.
00:21:16.24	And that's shown here.
00:21:19.03	So, MAT...
00:21:22.10	PCR analysis showed that the Rad51 protein was easily visible by a half an hour
00:21:28.10	after we initiated the double-strand break.
00:21:30.20	But you don't see Rad51 bound to the HML locus until considerably later.
00:21:35.20	So, the search to find the HML locus and to be able to facilitate this kind of
00:21:42.28	repair event is a time-dependent process.
00:21:46.14	And we could see that by using chromatin immunoprecipitation.
00:21:52.02	So again, just to review what we've said so far, there's a break.
00:21:56.10	The break gets resected.
00:21:58.14	The Rad51 protein, with the aid of Rad52 and other helper proteins, loads onto the single-stranded DNA,
00:22:06.16	initiates a search across the genome in order to be able to find its partner,
00:22:12.05	makes those new Watson-Crick base pairs, and now the next step in the process would be to
00:22:17.26	initiate new DNA synthesis to be able to copy the new sequences that are going to be used to
00:22:23.11	replace the sequences originally at the MAT locus.
00:22:27.22	And we worked out a way to see the new DNA synthesis.
00:22:33.27	So, here, what we realized is that if we had a pair of PCR primers, one of which is specific
00:22:40.15	for the donor and one of which is specific for the MAT locus,
00:22:44.08	they would originally be 200 kilobases apart.
00:22:47.22	And there would be no possibility of getting a PCR product.
00:22:51.04	But once the strand invasion has taken place, and just 50 base pairs of new DNA synthesis
00:22:56.22	has happened, now there's a covalent piece of DNA which will link these two primers together.
00:23:03.04	And therefore you could see the appearance of this PCR product as an indication of...
00:23:08.05	when had new DNA synthesis occurred?
00:23:10.24	As you can see by just looking at this... at this gel.
00:23:14.17	So, we could figure out when exactly this primer extension
00:23:20.24	-- the recruitment and use of DNA polymerase -- had taken place.
00:23:27.21	Here, we got another surprise.
00:23:29.28	And that is that the Rad54 protein, which is another chromatin remodeling protein,
00:23:35.21	turns out not to be able to do this step.
00:23:39.06	And so, even though Rad54 was not needed to do this initial strand invasion step,
00:23:44.11	as I showed you before, it turns out to be critical for doing this next step,
00:23:48.17	which is the primer extension.
00:23:50.06	And we think that there's some kind of rearrangement of the chromatin in the donor locus that requires
00:23:55.28	the Rad54 protein in order for the recruitment of new DNA polymerase to come, and to be able
00:24:01.28	to initiate this repair process.
00:24:04.28	So, if we do this in... sort of... with more time points than I've shown you in this illustration,
00:24:12.13	it takes about 10 minutes from the time we can see the double-strand break until
00:24:16.08	there's the recruitment of Rad51.
00:24:18.16	It takes at least 20 minutes after at that point... when the Rad51 filament can find
00:24:24.15	its partner, which is located 200 kilobases away on the same chromosome.
00:24:29.19	It takes a lot longer to find its partner when the partner is in another,
00:24:34.28	different chromosome.
00:24:36.08	And then after that, there's still another 20 minutes after this strand invasion
00:24:40.17	has taken place before the Rad51 protein can be gotten out of the way and the new DNA polymerase
00:24:47.08	has been recruited in order to initiate new DNA synthesis.
00:24:50.13	So, there's a whole series of relatively slow steps that we could identify as important
00:24:57.06	in this repair of a broken chromosome.
00:25:01.02	Okay.
00:25:03.07	I mentioned that repairing the break when the donor and the recipient are on the same chromosome
00:25:10.09	is faster than when the donor and the recipient are on different chromosomes.
00:25:15.16	And we can see that by using that PCR reaction... as to how much slower
00:25:21.06	the interchromosomal recombination event is relative to the intrachromosomal event.
00:25:27.18	But that's just one place.
00:25:29.02	I mean, we put a donor somewhere, and we asked, how did the repair take place?
00:25:34.16	But you'd like to know more globally, what does the position of the chromosome relative
00:25:39.18	to the donor have as a way of influencing this process?
00:25:45.06	And so here we took advantage of a work from Noble's lab, in which they used what is called
00:25:56.09	chromosome conformation capture, by using, again, formaldehyde cross-linking of DNA to DNA,
00:26:02.12	to ask, which pieces of DNA are more likely to be close together,
00:26:08.03	and which pieces of DNA are likely to be more far apart within the yeast nucleus?
00:26:11.22	And so, this is a cartoon of what... where all the chromosomes are inside the yeast nucleus.
00:26:17.24	We already knew from lots of cytology that all the centromeric regions are
00:26:24.07	clustered together at something called the spindle pole body, the centriole.
00:26:27.25	And so all those sequences are clustered together.
00:26:30.22	But then the question is, does it really matter if we make a break at this red sequence,
00:26:36.03	for example... does it matter whether the donor is here or here or here?
00:26:40.17	In other words, does this cartoon really predict how efficient recombination will be?
00:26:47.08	And so, to do this, we changed the system.
00:26:51.09	We put the HO cleavage site inside just another piece of DNA, not the original mating-type sequence,
00:26:57.04	which has now been deleted, but a gene called LEU2.
00:27:01.12	And then we placed a donor sequence that shares about two kilobases of homology
00:27:07.13	with the broken segment elsewhere in the genome.
00:27:10.00	And we just measured by viability how efficient was this repair event.
00:27:14.11	If it's very efficient, then the cells plated on galactose, where the HO endonuclease is induced,
00:27:20.03	should be very similar to what happens on glucose where all the cells can grow.
00:27:25.12	But if there... if repair is very difficult, then this number on galactose plates is going
00:27:31.00	to be a low number.
00:27:32.23	And so, to do this experiment, we put a double-strand break in one location, and then we made
00:27:38.08	20 strains where the donor was in many different locations.
00:27:41.09	And they were chosen in part because some of them were estimated to be close together,
00:27:46.25	and some of them were estimated to be far apart, relative to the site which is being broken.
00:27:53.06	And so, this is the result we got.
00:27:55.05	It turns out that the likelihood that two sequences are close together before
00:28:01.04	we induce double-strand break has a very strong effect on how efficient the repair event is.
00:28:06.28	Some of these events are 50 perfect efficient, and some of these events are a few percent efficient.
00:28:12.13	And that's all dependent on location.
00:28:15.00	It turns out, in mammals, the probability that those rearrangements
00:28:19.19	-- those translocations that I showed you at the very beginning, that you see in cancer cells --
00:28:25.11	is also very much indic... predicted by the proximity of the segments of DNA
00:28:30.25	that are undergoing rearrangements.
00:28:33.11	So, this seems to be a general feature of DNA repair, that the really slow step
00:28:40.27	in finding sequences to do the repair is searching for them over huge distances.
00:28:46.04	And the closer they are to begin with, the more efficient they are in doing this repair.
00:28:51.18	So, I mentioned previously that the Bloom's helicase plays an important role in the resolution
00:28:58.23	of some recombination structures, by being able to unwind double Holliday junctions
00:29:04.21	so that they don't end up being crossovers, and lead instead to non-crossover outcomes.
00:29:11.02	And the Bloom helicase carries this out in a... in a way that is very obvious from
00:29:18.28	this cytology, here.
00:29:20.15	Here, we see sister chromatids undergoing frequent exchange events, crossover events,
00:29:28.28	compared to the wild type.
00:29:31.08	Because this process of dissolving intermediates is incredibly efficient in mitotic cells.
00:29:40.26	And we showed in Saccharomyces that this same Bloom's helicase -- its homolog, called Sgs1,
00:29:47.12	has a similar role.
00:29:49.06	So, again, using the mating-type switching system and the inducible HO endonuclease...
00:29:57.25	except here we placed a donor on a different chromosome.
00:30:01.08	This donor has a mutation so it can't be cleaved.
00:30:04.27	And the restriction sites are arranged so that if you see a crossover, you can see that
00:30:10.04	that product is different from the non-crossover.
00:30:12.21	So, in wild type, there are very, very few crossover events
00:30:16.22	associated with this double-strand break repair event.
00:30:19.22	But when we get rid of the Bloom's helicase, Sgs1, or the topoisomerase, you can see
00:30:25.10	a very large increase, almost a four-fold increase in the level of crossing over,
00:30:30.23	which is associated with their absence.
00:30:33.08	And so this dissolving pathway is preserved all the way from yeast to humans.
00:30:41.04	So, I've gone through a number of steps that we are able to visualize in real time
00:30:48.28	by using the yeast mating-type switching system and related systems, with an inducible site-specific nuclease,
00:30:55.03	to be able to monitor in real time these kinds of events.
00:30:59.20	We sort of grouped them all into something we called in vivo biochemistry.
00:31:04.06	And we could describe each of these events in some detail.
00:31:09.14	In the next video, I'm gonna talk about another consequence of doing this kind of work,
00:31:15.21	which is to reveal how mutagenic this supposedly accurate repair event actually is.
00:31:22.18	It turns out that, compared to normal DNA replication, repair by these kinds of mechanisms
00:31:28.13	turns out to be almost 1000 times more mutagenic than a normal replicative event.
00:31:35.07	So, even though repairing in this way prevents the kinds of crazy translocations that I showed you
00:31:40.07	at the very beginning, it is not without risk.
00:31:43.27	And it turns out to be a reflection of the fact that this repair mechanism is
00:31:48.11	not using the same machinery, in detail, as the normal replication process.
00:31:53.08	So, all of this work that I've talked about was of course not just done... or, in fact,
00:31:59.03	very little of it done by me.
00:32:01.08	It was done by a huge number of people in my lab.
00:32:03.26	This is not a picture of my current lab, but rather a reunion of a large number of people
00:32:09.17	who have worked in my lab.
00:32:11.24	This slide just lists all the undergraduates, graduate students, and postdocs who have
00:32:18.04	worked in my lab over a very long career in order to learn a lot of the things that I've told you.
00:32:24.23	But in addition to these many people with... who have been in my lab, we also have collaborated
00:32:30.22	with an astonishing number of other labs in order to accomplish this work.
00:32:35.11	And these are simply listing all of the principal investigators at different labs.
00:32:39.24	Of course, they too had students and postdocs who were part of this process.
00:32:44.26	So, learning all of this material took quite a lot of effort on the part of many labs
00:32:51.00	to really define what we know about double-strand break repair.
00:32:54.14	Thanks very much.