Published online 4 December 2003

Repairing a double-strand chromosome break by : revisiting Robin Holliday’s model

James E. Haber*, Gregorz Ira, Anna Malkova and Neal Sugawara Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast , one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT ) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be perfor- med in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday’s model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking. Keywords: homologous recombination; yeast mating-type switching; Robin Holliday, DNA repair

1. INTRODUCTION the models we continue to discuss are fundamentally based on his idea that strand exchange creates both hetero- The model of Robin Holliday (1964), designed to explain duplex DNA and HJs. the major events in meiotic recombination—crossing-over, Our present understanding of recombination at the gene conversion and post-meiotic segregation—estab- molecular level is anchored in the relatively recent knowl- lished a conceptual framework that has guided nearly edge that meiotic recombination in eukaryotes is initiated 40 years of study. The model of Holliday (1964) envi- not by single-strand nicks, as envisioned by Holliday, but sioned that crossing-over began with a coordinated pair of by DSBs, created by a special topoisomerase VI-related single-strand nicks on homologous chromosomes followed enzyme, Spo11 (Sun et al. 1989; Bergerat et al. 1997; by a displacement and exchange of single strands (figure Keeney et al. 1997). DSBs were also detected in the best- 1). This led to the creation of the four-stranded structure studied example of recombination in mitotic cells, the we now call an HJ, which could be resolved to give both switching of MAT genes (Strathern et al. 1982). However, crossover and noncrossover outcomes. Mismatch repair of the first articulation of the idea that recombination was heteroduplex DNA could produce aberrant ratios of initiated by a DSB, was presented by Resnick (1976), alleles among the progeny. The model of Holliday (1964) based on the repair of chromosome breaks created by ion- accounted for the important genetic observations that had izing radiation. A similar, but more elaborated model, been made predominantly in of fruitflies and presented by Szostak, Orr-Weaver, Rothstein and Stahl fungi, as well as provocative observations coming from (1983), gained wide acceptance a few years later. This studies of bacteriophage and bacteria. The model could DSB repair model (figure 2a) was based primarily on explain why genetic exchange often involved formation of studies of transformation and gene targeting in budding regions of heteroduplex DNA and why gene conversions— yeast (Orr & Szostak 1983; Rothstein 1983), but it pro- the nonreciprocal transfers of genetic information from vided explanations for many observations that had not one homologous chromosome to another—often occurred been accounted for by the model of Holliday (1964) or by with an exchange of flanking genetic markers. Almost its successor, the single-strand nick model of Meselson & 40 years later, although many details of the model of Hol- Radding (1975). In this model, recombination is initiated liday (1964) have not withstood more recent discoveries, by ssDNA that was created by 5Ј to 3Ј exonucleases resecting the ends of the DSB. Strand invasion is made possible by the RecA/Rad51 strand invasion protein that * Author for correspondence ([email protected]). forms a filament on the ssDNA and then synapses with a One contribution of 18 to a Discussion Meeting Issue ‘Replicating and homologous donor sequence. The invasion of both ends reshaping DNA: a celebration of the jubilee of the double helix’. of the DSB leads to the formation of a pair of HJs. New

Phil. Trans. R. Soc. Lond. B (2004) 359, 79–86 79  2003 The Royal Society DOI 10.1098/rstb.2003.1367 80 J. E. Haber and others Revisiting Robin Holliday’s model

DNA synthesis, primed from the 3Ј OH ends of the invad- appearance of noncrossovers was coincident with the ing strands, is used to fill in any gaps. Then an HJ resol- appearance of double HJs and that crossovers did not arise vase is imagined to cleave the two HJs, sometimes yielding for another hour. This argues strongly that crossovers and crossovers, depending on how the junctions are cleaved. noncrossovers do not emerge as alternative outcomes from Support for this model came from physical analysis of a common intermediate, as the Szostak et al. or Holliday recombination intermediates, both in yeast meiosis and in models would imagine. Moreover, Allers and Lichten parallel studies of DSB-initiated recombination in mitotic showed that a significant proportion of double HJs were cells. First, 5Ј to 3Ј resection of DSB ends was shown both located on one side of the DSB, an outcome that is pre- in mitotic (White & Haber 1990) and meiotic (Sun et al. dicted for some SDSA models but not by the Szostak et 1991) cells; then Schwacha and Kleckner demonstrated al. model. These data make a strong case for several DSB- that fully ligated double HJs were in fact present during mediated recombination processes occurring at the same meiotic recombination (Schwacha & Kleckner 1995). time. How events are channelled into one pathway or the As originally conceived, the Szostak et al. (1983) model other(s) is not yet clear. imagined little role for mismatch repair, imagining that In parallel to the analysis of meiotic recombination are most gene conversions arose from the repair of gaps in the studies of mitotic recombination, many of which have broken chromosome. However, subsequent studies, using been done using a galactose-inducible site-specific HO mismatch repair-defective alleles and mismatch repair endonuclease to create a single DSB, repaired by homolo- mutants (White et al. 1985; Williamson et al. 1985), as gous recombination (reviewed by Haber (2002)). HO well as mapping the ends of Spo11-generated DSBs (Sun endonuclease evolved to catalyse the switching of budding et al. 1991), made it clear that there are long heteroduplex yeast MAT genes, in which the MATa-orMAT␣-specific regions formed between the recipient and donor DNA DNA sequences (Ya or Y␣) are replaced by a gene con- sequences and that most gene conversions come from mis- version event using one of two donor sequences, HML␣ or match repair. HMRa, located at opposite ends of the same chromosome In the next decade it became apparent that there was a harbouring the MAT locus (figure 3a). A galactose- need to modify the picture still further. From studies of induced HO gene (Jensen & Herskowitz 1984) made it budding yeast MAT gene switching (Nasmyth 1982; possible to induce a DSB in virtually all cells in a McGill et al. 1989), transposable element recombination synchronous fashion, so that one can follow the kinetics in Drosophila (Gloor et al. 1991) and from transformation of switching on Southern blots (Connolly et al. 1988; fig- experiments in mammalian cells (Belmaaza & Chartrand ure 3b). Resection of DNA ends occurs at ca. 4 kilobase 1994), an alternative set of recombination models (kb) hϪ1, leaving 3Ј ends (White & Haber 1990; Fishman- emerged, known as SDSA mechanisms (figure 2bϪd). Lobell et al. 1992). PCR can be used to identify an inter- Here, strand invasion was regarded as rate limiting so that mediate of recombination, the beginnings of new DNA one end would succeed while the second end remained synthesis after strand invasion; this occurs ca. 20–30 min unengaged. Moreover, new DNA synthesis was imagined after the DSB can be detected and 30 min before MAT to be more like transcription than normal semi-conserva- switching is complete (White & Haber 1990). Similar tive replication, so that the newly synthesized strand would analyses can be done at other loci, by inserting a cloned be displaced. When the displaced strand overlapped the HO recognition site at another chromosomal location. second end, annealing would occur and the second 3Ј end would be used to copy the second strand from the new 2. STUDYING DNA REPAIR WITH CONDITIONAL template. A key difference between this mechanism and MUTATIONS IN DNA REPLICATION the DSB repair model of Szostak et al. is that all the newly synthesized DNA would be found in the recipient locus We have made extensive use of mutations in genes and that all the events would be found as noncrossovers, involved in DNA replication and repair to assess their as there are no stable double or single HJs. Some studies roles in gene conversion. Many of the genes in which we of gene conversion in yeast meiosis appear to agree with are interested are essential, but we can arrest cells carrying the predictions of SDSA models (Porter et al. 1993; Gil- conditional-lethal mutations (high- or low-temperature bertson & Stahl 1996), as do many results from mitotic sensitive mutations) at their restrictive temperature before yeast cells, discussed more in this and the following sec- inducing HO endonuclease from its galactose-inducible tion. However, it is possible to imagine several ways in promoter. DNA can be monitored at the restrictive tem- which the SDSA mechanism could be modified to allow perature, without returning cells to growth conditions. the formation of double HJs and produce crossovers as This ‘in vivo biochemistry’ approach has revealed some well as noncrossovers (Ferguson & Holloman 1996; surprising aspects of DSB-induced recombination. Most Paˆques & Haber 1999; Allers & Lichten 2001b), one of strikingly, nearly all of the components of normal DNA which is shown in figure 2e. In such variations, the pos- replication appear to be required to carry out MAT ition of the double HJ would probably be on one side of switching, where no more than 700 base pairs (bp) of new the DSB, rather than flanking it, as in the Szostak et al. DNA synthesis appears to be required. The clamp, model. PCNA, and its clamp loader, RFC, are all required, and In fact, there are apparently several mechanisms of mutations in both Pol␦ and Pol⑀ severely delay and impair recombination competing for the attention of the DSB. completion of recombination (Holmes & Haber 1999). Allers & Lichten (2001a) found that a ndt80 mutation pre- But most striking is that Pol␣ and primase, along with the venting transcription of late meiotic genes in budding Okazaki-processing FEN-1 enzyme, Rad27, are all yeast allowed noncrossovers but not crossovers to be com- required (Holmes & Haber 1999). An example, showing pleted. Supporting this finding, they showed that the the failure of MAT switching at the restrictive temperature

Phil. Trans. R. Soc. Lond. B (2004) Revisiting Robin Holliday’s model J. E. Haber and others 81

Figure 1. The model of Holliday (1964) for gene conversion and associated crossing-over. Coordinate single-strand nicks and strand exchange produce two regions of heteroduplex strand exchange and DNA and the formation of a four-strand DNA strand heteroduplex DNA formation exchange junction, now known as the HJ. Subsequent mismatch repair can either restore or convert heteroduplex, resulting in various types of gene conversion outcome. One example is shown, where heteroduplex is repaired in each HJ resolution case in favour of the red strand. When mismatches are not repaired, subsequent DNA replication will reveal the heteroduplex as a sectored colony, known as post-meiotic segregation in meiosis. mismatch repair

DSB

(a) DSB repair model (b) SDSA (c) leading/lagging (d) modified SDSA, (e) SDSA converted to of Szostak et al. SDSA by Allers & Lichten a double HJ (2001b)

Figure 2. Models of homologous recombination initiated by a DSB. Only noncrossover outcomes are shown, but crossovers can be obtained even from SDSA mechanisms if the D-loop is ‘captured’ by the second end. Note that in SDSA (e.g. b, c and d), nearly all the newly synthesized DNA (light blue) will be in one of the two recombinant molecules, whereas in the Szostak et al. model, both recombined molecules will have one strand of newly synthesized DNA.

(37 °C) of a temperature-sensitive mutation of primase effect of inactivating Pri2, but when the sequences are in (pri2-1) is shown in figure 4a. Primase and Pol␣ are inverted orientation and repaired by gene conversion, pri2- components of lagging-strand synthesis and should not be 1 prevents repair at 37 °C (figure 4b). needed if the invading 3Ј ends of the DSB act as primers, One reason that we like the idea that there is a modified either in the Szostak et al. model or in the standard version replication fork required for ‘gap repair’ is that we imagine of SDSA. This result raises the possibility that gene con- a similar type of fork is needed to carry out BIR, which version uses a modified replication fork. It appears that would begin with the same strand invasion step (figure 6). the failure of MAT switching at 37 °Cinpri2-1 is not This mechanism has its roots in studies of phage T4 late caused by tying-up all the DNA replication machinery in replication, which was seen to be a recombination-depen- stalled replication forks, because similar results were dent replication mechanism. Skalka (1974) provided a found in G1-blocked cells (Holmes & Haber 1999), where molecular conception that has not changed significantly. no DNA replication occurs. However, because G1- In Escherichia coli, such recombination-dependent DNA arrested cells also have reduced recombination, we con- replication was invoked to explain origin-independent tinue to investigate this question. DNA synthesis (Kogoma 1997). More recently, it has We have also attempted to rule out that primase is been suggested as a way in which broken replication forks needed to initiate copying from a 3Ј end of an invading might be re-started, both in bacteria and in eukaryotes strand by examining the process of SSA, where it would (Haber 1999; Michel 2000; Rothstein et al. 2000; Kraus be difficult to imagine a role for lagging-strand synthesis et al. 2001). in filling-in gaps in DNA to complete the process (figure During transformation, BIR may lead to the copying 4c). Using a plasmid containing two homologous of extensive regions of a chromosome; in fact it has been sequences, one of which carries an HO cut site, we suggested that in E. coli a transforming fragment may copy showed that when these sequences are in direct orientation an entire chromosome. Evidence of ‘copy-choice’ DNA and repair occurs almost completely by SSA there is no synthesis during phage recombination has also been

Phil. Trans. R. Soc. Lond. B (2004) 82 J. E. Haber and others Revisiting Robin Holliday’s model

(a) RE

HMLα Yα EL IL a1 Ya ER IR MATa Ya HMRa

HO endonuclease

MATα α2 α1 Yα

(b) 0 1 2 4 hours

MATα

MATa HO cut

Figure 3. Physical monitoring of yeast MAT switching. (a) Chromosome III of budding yeast contains the MAT locus, plus two distant, unexpressed copies of mating-type information at HML and HMR. HML and HMR are kept silent by deacetylation of highly positioned nucleosomes, organized by two cis-acting silencer sequences, E and I. (b) Southern blot analysis of DNA digested with StyI restriction endonuclease, which cleaves a site within Ya but not in Y␣, thereby producing a distinctive restriction fragment that shows when MATa is replaced by MAT␣. In the example shown, both HML and HMR carry Y␣, so that MATa switches only to MAT␣. MATa by a galactose-induced HO endonuclease is seen within 30 min of induction, but appearance of the MAT␣ product does not occur for at least another hour. reported (Kuzminov & Stahl 1999; Motamedi et al. 1999). 2002). In budding yeast, there are two RAD52-dependent A similar result has been obtained in yeast, where Morrow BIR pathways that can maintain telomeres in different et al. (1997) showed that a small centromere-containing ways (Lundblad & Blackburn 1993; Le et al. 1999; Teng DNA fragment, with the same sequences at both ends et al. 2000). One pathway involves Rad51, Rad54, Rad55 could recombine with a homologous sequence on a chro- and Rad57; the other needs Rad50-Mre11-Xrs2 and mosome, leading to the formation of a new chromosome Rad59 (figure 7). We have also shown that there are two with two identical chromosome arms. Bosco & Haber BIR pathways for repairing a single DSB in a diploid (1998) then showed that cutting off the end of a chromo- (Malkova et al. 1996; Signon et al. 2001; also A. Malkova, some by HO endonuclease led to the formation of a non- M. Naylor and J. E. Haber, unpublished data). The two reciprocal translocation, using only 70 bp of homology pathways have the same genetic requirements as does telo- adjacent to the DSB as the means to invade another chro- mere repair without telomerase: one is RAD51 dependent mosome arm and initiate replication to the telomere. and the other is RAD50 and RAD59 dependent. We have In eukaryotes, BIR may be particularly important at tel- argued, based on plasmid intrachromosomal recombi- omeres, whose integrity can be maintained in the absence nation model systems, where both gene conversion and of telomerase by recombination-dependent DNA repli- BIR can occur, that the Rad51 pathway becomes inef- cation (i.e. BIR). In mammalian cells, such processes are ficient when homology is less than 70–100 bp, whereas the termed alternative lengthening of telomeres (ALTs). ALT Rad59- and Rad50-dependent pathway, which appears to also seems to depend on recombination, although genetic be only BIR (followed by SSA) is remarkably efficient, requirements have not been established (Henson et al. even with as few as 30–33 bp homology on either side of

Phil. Trans. R. Soc. Lond. B (2004) Revisiting Robin Holliday’s model J. E. Haber and others 83

(a) 012345 0 1 2 3 4 5 hours

MATα

MATa

HO cut

23 ˚C 37 ˚C

(b)(c) 012345 0 1 2 3 4 5 hours 0 1.5 6 0 1.5 6 hours co co gc gc parental gc gc HO cut co co

HO cut

deletion HO cut 23 ˚C 37 ˚C 23 ˚C 37 ˚C

Figure 4. Effect of a temperature-sensitive primase mutation on DSB repair. (a) Cells carrying a temperature-sensitive allele of primase (pri2-1) are able to switch from MAT␣ to MATa at the permissive temperature, 23 °C, but are severely impaired in switching at the restrictive temperature of 37 °C (data from Holmes & Haber 1999). (b) A plasmid with two inverted copies of E. coli LacZ sequences (Fishman-Lobell et al. 1992; Ira & Haber 2002), one of which carries an HO cleavage site (arrow) was analysed in the pri2-1 strain (Holmes & Haber 1999). Repair, which can occur both by gene conversion and by break-induced replication followed by SSA (Ira & Haber 2002), is impaired at 37 °C. Restriction fragments from gene conversion without crossing-over (gc) and gene conversion with crossing-over (co) are shown. (c) By contrast, a similar plasmid, but with the repeated sequences in direct orientation, can complete repair by SSA even at the restrictive temperature for pri2-1. a DSB (Ira & Haber 2002). In fact, Rad51 becomes longer homology. Type II survivors elongate telomere inhibitory when homology is so short; deleting Rad51 sequences themselves. Because yeast telomeres are dramatically increases the Rad59–Rad50- pathway irregular TG1–3 sequences, there are likely to be only rela- efficiency. These findings lead us to suggest how the two tively short regions of perfect homology between one telomere-maintenance pathways operate. Type I survivors resected chromosome end and template sequences on lacking telomerase proliferate by recombining onto nearly another telomere (or a looped-back region of the same all deteriorating ends long ca. 6 kb repeats and other sub- telomere). telomeric regions that are shared at all chromosome ends. Still, the repair replication fork involved in gene conver- This is done by the Rad51-dependent pathway that needs sion appears to differ in several important respects from

Phil. Trans. R. Soc. Lond. B (2004) 84 J. E. Haber and others Revisiting Robin Holliday’s model

wild-type RPA70-L45E

est2 or tlc1 strains lacking telomerase MATα type I: BIR using long homology in subtelomeric regions MATa HO cut RAD52 and RAD51, RAD54, RAD55, RAD57 dependent 0 0.5 1 2 3 4 5 0 0.5 1 2 3 4 5 hours

Figure 5. A replication-competent mutation of the largest subunit of RPA (rfa1-t11) is defective in HO-induced gene type II: BIR using short homology conversion of MAT. Whereas wild-type cells switch in irregular TG1–3 repeats efficiently from MATa to MAT␣, cells carrying the rfa1-t11 mutation have severely reduced switching.

RAD52 and RAD50, MRE11, XRS2, RAD59 dependent

Figure 7. Telomere maintenance in the absence of telomerase. Yeast telomeres (vertically hatched lines) shorten and eventually lose their end-protection when telomerase RNA or protein components are deleted. After many generations, cells senesce, but a small proportion of survivors arise. Two types of survivor, with different genetic requirements, have been identified. Type I survivors proliferate long subtelomeric regions including YЈ elements to nearly all chromosome ends, although small amounts of telomere sequence are retained at these ends. Type II survivors manage to elongate telomere sequences themselves. Both types of survivors are eliminated in the absence of RAD52, but type I requires RAD51, RAD54, RAD55 and RAD57, whereas type II requires RAD50, MRE11, XRS2 and RAD59. Type II survivors also require the Sgs1 helicase, although this requirement is not seen when BIR is initiated in other chromosome contexts.

can also occur in nocodazole-blocked cells when Mcm proteins are believed to be expelled from the nucleus? Figure 6. BIR: this occurs when one end of a DSB can What does RPA do in recombination, i.e. in establishing invade into an intact template sequence and set up a repair replication, that is distinct from a role in replication itself? replication fork that can copy DNA to the end of a chromosome arm. 3. ROLE OF MISMATCH REPAIR the normal replication fork. Normal replication depends The third key element of the model of Holliday (1964) on the continuous activity of Mcm proteins that are was that gene conversions arose by mismatch correction believed to act as a helicase at the replication fork (Labib of heteroduplex DNA. In the model of Holliday (1964), et al. 2000). We have used the temperature-sensitive mismatch correction was a late step, perhaps after resol- mutations that block both initiation and elongation of nor- ution of the HJ. However, during MAT switching, it mal replication (Labib et al. 2000) and discovered that appears that mismatch correction occurs early in the pro- MAT switching can occur at the restrictive temperature of cess. This was shown by following a mismatch that would both mcm4 and mcm7 mutants (X. Wang and J. E. Haber, be formed between the invading ssDNA from MAT␣ and unpublished data). Another mutation that reveals differ- the donor HMRa-stk allele (Haber et al. 1993). The ences in recombination and repair is a the L45E mutation stk/wild-type mismatch (A/A) lies only eight bases from of the large subunit of the ssDNA binding protein, RPA, the 3Ј end of the invading single strand. Once the 3Ј end known as rfa1-t11. This mutation has no effect on normal of the invading strand begins to be elongated by new DNA replication, but severely inhibits MAT switching (figure synthesis, it is possible to PCR amplify this intermediate, 5) and SSA (Umezu et al. 1998). These data raise many using primers specific for the Ya region of HMR and for questions: what helicase, if any, is used in place of the sequences just distal to MAT. As soon as the intermediate Mcm complex? Are Mcm proteins needed for BIR, which can be amplified, and 30 min before final product can be

Phil. Trans. R. Soc. Lond. B (2004) Revisiting Robin Holliday’s model J. E. Haber and others 85

Ya stk ways in which Robin Holliday’s prescient model still HMRa-stk describes many of the steps we continue to investigate: strand invasion and heteroduplex formation, resolution of MATα HJs and mismatch repair. Still, what goes on inside the Yα Rad51/RecA filament remains only vaguely described and ΗΟ the ways in which many additional proteins aid in this pro- cess are also only fleetingly glimpsed. By the time of the stk Diamond Jubilee of solving DNA’s structure (2028), per- HMRa-stk haps all these mysteries will have been solved. MATα Work in the Haber laboratory has been the collaborative effort of many students and post-docs. In addition to the coauthors, recent work has been done by M. Vaze and X. Wang. Funda- mentally important contributions were made by M. Colaia´- stk covo, B. Connolly, J. Fishman-Lobell, A. Holmes, F. Paˆques, HMRa-stk B. Ray, G.-F. Richard, N. Rudin and C. White. The MAT MAT switching system in yeast depended on the pioneering contri- butions of I. Takano and Y. Oshima, J. Hicks, J. Strathern, I. Ya Herskowitz, A. Klar, K. Nasmyth, F. Heffron and R. Kos- triken. Research in the Haber laboratory has been supported by grants from the National Institutes of Health, the National Science Foundation, the Department of Energy, the American Cancer Society and the Leukemia and Lymphoma Society of America.

MAT-distal REFERENCES

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