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Repairing a Double-Strand Chromosome Break by Homologous Recombination: Revisiting Robin Holliday’S Model

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Repairing a Double-Strand Chromosome Break by Homologous Recombination: Revisiting Robin Holliday’S Model Published online 4 December 2003 Repairing a double-strand chromosome break by homologous recombination: 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 Saccharomyces cerevisiae, 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 meiosis 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.
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