Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity

Autor: Jennifer A. Clikeman, Kimberly S. Paffett, Or Amit, Mark A. Brenneman, Rosa T. Sterk, Jac A. Nickoloff, Yi-Chen Lo
Rok vydání: 2006
Předmět:
Zdroj: Molecular and cellular biology. 26(11)
ISSN: 0270-7306
Popis: The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for maintaining genome stability and cancer suppression. DSBs are produced by ionizing radiation, genotoxic chemicals, and nucleases and when replication forks encounter DNA damage. Broken ends are converted to Rad51 nucleoprotein filaments that search for and invade homologous duplex DNA, producing a Holliday junction (HJ) intermediate. Branch migration of HJs extends or eliminates heteroduplex DNA (hDNA), and mismatches in hDNA are repaired, resulting in a region of localized loss of heterozygosity termed a gene conversion tract. Crossovers accompany some gene conversions, posing risks of deletions, inversions, translocations, and large-scale loss of heterozygosity (31, 45, 54). The mechanisms that suppress tract lengths and crossovers are important to elucidate because they determine the extent and frequency of the loss of heterozygosity during DSB repair by HR and thereby regulate genome stability. In the yeast Saccharomyces cerevisiae, mitotic and meiotic crossovers are suppressed by Sgs1 (26, 55), a member of the RecQ helicase family that includes five human proteins, three of which (BLM, WRN, and RECQ4) suppress tumors (25). RecQ helicases have conserved structures and interactions with type I topoisomerases (e.g., yeast Top3). Yeast Rmi1 is in complex with Sgs1-Top3 and may promote binding to branched DNAs or Top3 strand passage (10, 43). Yeast sgs1, top3, and rmi1 mutants show DNA damage hypersensitivity, genome instability, slow growth, poor sporulation, and hyperrecombination (10, 43). Sgs1-Top3-Rmi1 has roles in processing HR intermediates, restarting blocked or collapsed replication forks, and activating S-phase checkpoint arrest (5, 6, 8, 10, 13, 15, 16, 19, 26, 32, 43, 48, 55). These roles in replication are thought to underlie the synthetic lethality/sickness of sgs1 with srs2, rrm3, slx1, slx4, mus81, and mms4 mutations (15, 17, 58, 64). The synthetic lethality/sickness of sgs1Δ srs2Δ mutants is suppressed by defects in RAD51 and other HR genes, suggesting that Sgs1 and Srs2 play redundant roles in resolving recombination intermediates that arise during replication (15). sgs1Δ cells are hypersensitive to DNA-damaging agents like methyl methanesulfonate (MMS) and to replication stress induced by hydroxyurea (7, 16, 40, 56), and they show enhanced rates of spontaneous HR, gross chromosomal rearrangements, and chromosome loss (3, 20, 44, 68). Cells lacking Sgs1 or Srs2 frequently arrest as large-budded cells with a single nucleus in the mother cell or “stuck” between mother and daughter cells (37). Thus, Sgs1 has important roles in both DNA repair and replication. Crossover suppression by Sgs1 suggests that this protein promotes HJ resolution by a noncrossover mechanism, and this suggestion is supported by biochemical evidence. RecQ, Sgs1, and BLM each display DNA binding and ATP-dependent helicase activity and have a strong affinity for four-way junctions that resemble HJs (5, 24, 28, 74). Moreover, RecQ, Sgs1, and BLM branch migrate HJs (5, 24, 28) and BLM-Top3α resolves a synthetic double-HJ substrate by “dissolving” HJs into one another via reverse branch migration; this produces a hemicatenane that is unlinked by Top3α (74). HJ branch migration by BLM and the dissolution of double HJs by BLM-Top3α are both ATP dependent, presumably reflecting a requirement for BLM helicase activity (28, 74). Although the Sgs1 unwinding of duplex DNA requires ATP and a functional helicase/ATPase domain (6, 34), ATP/helicase dependencies of Sgs1 in HJ branch migration have not been reported. A reversed replication fork resembles an HJ, hence Sgs1-Top3-Rmi1 may process related branched DNA structures that arise at blocked replication forks and during DSB repair by HR (39). Ira et al. (26) suggested that Sgs1 suppresses crossovers by reverse branch migrating double HJs until they converge and are resolved without crossing over by Top3. This model makes two testable predictions. First, in the absence of Sgs1, double HJs would be free to forward branch migrate, extending hDNA and increasing conversion tract lengths. This prediction is consistent with more frequent association of crossovers with long conversion tracts (27, 62). Second, reverse branch migration should require a functional Sgs1 helicase, therefore tract lengths should also be increased in helicase-defective mutants. Consistent with the first prediction, we confirmed that crossovers are increased in sgs1Δ and show that allelic gene conversion tract lengths are increased. Surprisingly, these phenotypes were suppressed by helicase-defective Sgs1, thus defining two new helicase-independent roles for Sgs1. We also define three new helicase-dependent roles for Sgs1 in suppressing DSB-induced chromosome loss, chromosome missegregation, and synthetic lethality in srs2Δ. These results are discussed in relation to Sgs1 function in replication and HR-mediated DSB repair.
Databáze: OpenAIRE
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