Initiation and Completion of Spontaneous Mitotic Recombination Occur in Different Cell Cycle Phases

COMMENTARY

Initiation and completion of spontaneous mitotic recombination occur in different cell cycle phases

Lorraine S. Symington1 Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032

itotic recombination is re- quired to repair DNA double- M strand breaks (DSBs), to restart stalled/collapsed replication forks, and functions as an alterna- tive mechanism to elongate telomeres in cells lacking telomerase. Although these functions preserve genome integrity, mitotic recombination can also cause loss of heterozygosity in cells with polymorphic chromosomes and lead to gross chromosome rearrangements if it occurs between dispersed repeats. Much of our un- derstanding of recombination mechanisms is based on studies in fungi in which all of the products of an individual meiotic recombination event can be recovered. Two types of recombination events have been identiﬁed during meiosis: crossing over and gene conversion. A crossover between linked heterozygous markers results in new linkage arrangements, but the markers still display 2:2 segregation. By contrast, gene

conversion represents the nonreciprocal Fig. 1. Gene conversion events associated with crossing over following G1-orG2-induced DSBs. A DSB in G1 transfer of information between two ho- is replicated, and both sister chromatids are repaired using the homolog nonsisters as templates; alter- mologous sequences where one allele is natively, one broken chromatid might repair first and then be used to template repair of the other broken duplicated and the other is lost, resulting sister chromatid. Repair of the breaks is accompanied by transfer of polymorphic markers from the un- in a 3:1 segregation for heterozygous mark- damaged to the broken chromatid, resulting in gene conversion (boxed area). Only one of the two repair ers in the spore colonies. About 50% of events is associated with an RCO. If the same polymorphic markers are converted during both repair events, meiotic conversions are associated with then a 4:0 tract will result; if one repair event involves more markers, a hybrid 4:0/3:1 tract will be formed. A G2 DSB would be expected to give rise to a RCO with no detectable gene conversion if the conversion tract is crossing over, suggesting that these pro- very short or to a 3:1 conversion. The homologs are shown in dark and light blue, respectively. The poly- cesses are mechanistically linked. Because morphic sites are indicated by small solid circles, and the open circles represent the centromeres. mitotic recombination is much less frequent than during meiosis, events are usually selected and typically only one of the two that all of the chromatids engaged in the over to generate the sectored colony di- daughter cells produced following re- recombination event are recovered and, agnostic of an RCO. combination is recovered. Thus, little in- by scoring the presence of the heterozy- To test this hypothesis, Lee and Petes (1) formation exists on the mechanism, the gous markers in the two halves of the treated diploid cells synchronized in the time in the cell cycle when recombination sectored colony, knowledge of the mech- G1 or G2 stages of the cell cycle with ioniz- occurs, and the nature of the initiating le- anism can be derived. Two surprising re- ing radiation (IR) and then analyzed the sion(s). Using a clever genetic assay that sults emerged from these studies. First, spectrum of recombination events in the enables recovery of both products of a re- conversion tracts associated with sponta- red/white sectored colonies. The conver- ciprocal crossover event (RCO), Lee and neous mitotic crossovers were much lon- sion events in the G -irradiated cells were Petes (1) provide compelling evidence that 1 ger than meiotic conversion tracts. remarkably similar to the events observed spontaneous mitotic RCOs in G2 cells re- Second, some conversion tracts showed spontaneously, with 4:0 and 3:1/4:0 hybrid sult from a DSB present on one chromo- a 4:0 segregation of the markers or showed conversion tracts representing about 40% some before DNA synthesis. hybrid tracts with some markers segregat- In previous studies Barbera and Petes ing 4:0 adjacent to markers segregating of the total events. In contrast, only simple crossovers (no associated conversion) and (2) described a genetic assay that allows 3:1. Although gene conversion in a G1 3:1 conversion tracts were recovered from selection of both products of a rare G2 cell could give rise to 4:0 segregations, the G -irradiated cells; no 4:0 or 3:1/4:0 mitotic RCO in the form of red/white a crossover in G1 would not generate 2 sectored colonies. In a refinement of this a sectored colony. Thus, one possible ex- hybrid tracts were detected. Furthermore, assay using haploid parents with 0.5% se- planation for the 4:0 and hybrid tracts the median conversion tract lengths quence divergence, Lee et al. (3) were able is that a DSB present on one chromosome to map the position of RCOs within in a G1 cell is replicated, resulting in a 120-kb region of chromosome V and to two broken sister chromatids that are Author contributions: L.S.S. wrote the paper. detect marker conversions associated with repaired from the intact homolog non- The author declares no conflict of interest. the crossover (Fig. 1). Thus, this system sisters in G2. One of these repair events See companion article 10.1073/pnas.1001940107. is analogous to meiotic tetrad analysis in would have to be associated with a cross- 1E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1003050107 PNAS Early Edition | 1of2 Downloaded by guest on October 1, 2021 associated with RCOs from G1-irradiated Consistent with this idea, rare spontaneous combination genes are transcriptionally fi cells was 7.3 kb, not signi cantly different Rad52 foci that fail to resolve in G2 are regulated and not expressed during the G1 from the spontaneous conversion tract sometimes observed in G1 cells, suggesting phase of the cell cycle (12). G1 DSBs lengths (6.5 kb), but significantly longer that a cell with a broken chromosome do not activate the DNA damage check- than the G2 conversion tracts (2.7 kb). sometimes adapts and divides (5). point in yeast, cells initiate S-phase, and Consistent with the decrease in conversion replication forks progress with normal ki- tract length, more of the G2 RCOs have no These findings support netics in the presence of a DSB (13). The associated conversion, compared with the two broken chromatids resulting from G1-irradiated and spontaneous events. the idea that a DSB replication through the DSB then engage These findings support the idea that one or both nonsister chromatids to tem- a DSB present in G persists through 1 present in G1 persists plate repair (Fig. 1). These two repair S-phase and that the duplicated sister events could result in differing conver- chromatids, both harboring a DSB at the through S-phase. sion tract lengths, giving rise to the hybrid same position, are repaired in G2 (Fig. 1). 3:1/4:0 tracts observed for both spontane- A number of questions arise from these ous and G1-irradiated diploids. observations. First, what is the source of In principle, a G1 DSB in diploid cells can The mitotic conversion tracts associated be repaired by nonhomologous end joining spontaneous DSBs in G1 cells? Second, with spontaneous and G1 DSB-induced why do cells fail to repair the DSB in G1 (NHEJ) or by homologous recombination. RCOs are long and continuous (1, 3). and progress through S-phase with a bro- Although DSBs made by rare-cutting en- These could result from a long excision ken chromosome? Third, do gene con- donucleases are substrates for NHEJ, this tract by mismatch repair of an hDNA in- version events derive from a heteroduplex pathway functions poorly to repair IR-intermediate or by double-strand gap repair. Saccharomyces cerevisiae DNA (hDNA) precursor? duced lesions in Studies of DNA end resection have shown Spontaneous DSBs in G1 could result (6). However, NHEJ is the primary the preferential degradation of the 5′ from closely spaced excision repair inter- mechanism to repair IR-induced DSBs in strand and have demonstrated that the 3′ mediates or from the activity of topo- G1-phase mammalian cells (7). Thus, end remains intact for several hours. isomerases. Most spontaneous DSBs are replication of a G1 DSB and subsequent However, in the absence of repair, the 3′ thought to occur during S-phase, for exam- repair in G2 might be less frequent in end is lost as well (14, 15). In the time ple, when the replication fork encounters mammalian cells than budding yeast. Sev- between the induction of a DSB in G and a transient single-strand break on one of eral lines of evidence suggest that HR is 1 repair in G2, the ends could be resected the template strands resulting in replication suppressed in G1 cells. First, the resection more than 5 kb, resulting in long hDNA fork collapse. Collapsed forks are repaired of DSBs in haploid G1 cells is less exten- tracts, or both 5′ and 3′ ends could be by homologous recombination using the sive than observed in cycling or G2-ar- degraded, resulting in large gaps that rested cells and is activated by cyclin- partially replicated sister chromatid. DSBs would give rise to gene conversion without dependent kinase as cells progress through made by IR in G2 cells are also preferentially an hDNA intermediate. Analysis of re- repaired using the sister chromatid (4). S-phase (8, 9). The resection of DNA combination events in mismatch repair These events would go undetected in the Lee ends to generate 3′ single-strand DNA mutants using this genetic assay should and Petes (1) assay, which requires re- tails is necessary for Rad51 binding to address the question of whether an hDNA combination between homologs. It is possi- initiate homologous pairing and strand intermediate is involved. ble that a broken chromatid present in G2/M exchange (10). Second, Rad52, which is essential for HR in budding yeast, does that fails to repair using the sister or that is ACKNOWLEDGMENTS. Work in my laboratory on generated during mitosis might be segre- not associate with G1 DSBs to form de- recombination mechanisms is supported by Na- gated and progress to the next cell cycle. tectable foci (11). Third, several re- tional Institutes of Health Grant GM041784.

1. Lee PS, Petes TD (2010) Mitotic gene conversion events 6. Daley JM, Palmbos PL, Wu D, Wilson TE (2005) Non- 11. Lisby M, Rothstein R, Mortensen UH (2001) Rad52 induced in G1-synchronized yeast cells by gamma rays homologous end joining in yeast. Annu Rev Genet forms DNA repair and recombination centers during are similar to spontaneous conversion events. Proc Natl 39:431–451. S phase. Proc Natl Acad Sci USA 98:8276–8282. Acad Sci USA, 10.1073/pnas.1001940107. 7. Mahaney BL, Meek K, Lees-Miller SP (2009) Repair of 12. Basile G, Aker M, Mortimer RK (1992) Nucleotide se- 2. Barbera MA, Petes TD (2006) Selection and analysis of ionizing radiation-induced DNA double-strand breaks quence and transcriptional regulation of the yeast re- spontaneous reciprocalmitotic cross-overs inSaccharomy- by non-homologous end-joining. Biochem J 417: combinational repair gene RAD51. Mol Cell Biol 12: ces cerevisiae. Proc Natl Acad Sci USA 103:12819–12824. 3235–3246. 639–650. 3. Lee PS, et al. (2009) A fine-structure map of spontane- 13. Doksani Y, Bermejo R, Fiorani S, Haber JE, Foiani M 8. Aylon Y, Liefshitz B, Kupiec M (2004) The CDK regu- ous mitotic crossovers in the yeast Saccharomyces cer- (2009) Replicon dynamics, dormant origin firing, and lates repair of double-strand breaks by homologous evisiae. PLoS Genet 5:e1000410. terminal fork integrity after double-strand break for- 4. Kadyk LC, Hartwell LH (1992) Sister chromatids are pre- recombination during the cell cycle. EMBO J 23: mation. Cell 137:247–258. – ferred over homologs as substrates for recombina- 4868 4875. 14. Mimitou EP, Symington LS (2009) DNA end resection: tional repair in Saccharomyces cerevisiae. Genetics 9. Ira G, et al. (2004) DNA end resection, homologous re- Many nucleases make light work. DNA Repair (Amst) 8: 132:387–402. combination and DNA damage checkpoint activation 983–995. 5. Alvaro D, Lisby M, Rothstein R (2007) Genome-wide require CDK1. Nature 431:1011–1017. 15. Zierhut C, Diffley JF (2008) Break dosage, cell cycle analysis of Rad52 foci reveals diverse mechanisms im- 10. Krogh BO, Symington LS (2004) Recombination pro- stage and DNA replication influence DNA double pacting recombination. PLoS Genet 3:e228. teins in yeast. Annu Rev Genet 38:233–271. strand break response. EMBO J 27:1875–1885.

2of2 | www.pnas.org/cgi/doi/10.1073/pnas.1003050107 Symington Downloaded by guest on October 1, 2021