RAD1 and RAD10, but Not Other Excision Repair Genes, Are Required for Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

Abstract

HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1⌬ and rad10⌬ mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair genes and their human homologs in DSB-induced recombination and repair.Genes belonging to the epistasis group RAD3 are required for nucleotide excision repair (NER) of UV-damaged DNA in the yeast Saccharomyces cerevisiae (for a recent review, see reference 40). Some of these genes also function in other pathways of DNA metabolism. Genomic deletions of RAD3 (34) and SSL2 (RAD25) (17, 38) are lethal in haploid cells, indicating their essential role in maintaining cell viability. Experiments using conditional lethal mutations of RAD3 and SSL2 have demonstrated that their essential function is most likely their role in RNA polymerase II transcription (18,19,41). Substantiating the idea of an intimate linkage between NER and transcription were recent findings that protein products of at least RAD2, RAD3, RAD4, and SSL2 are components of or are physically associated with RNA polymerase II basal transcription factor b (5, 12).Experimental data also show that some NER genes have a role in mitotic recombination. Mutations in RAD1 and RAD10 reduce intrachromosomal recombination between directly repeated sequences and decrease the efficiency of homologous integration of linear DNA fragments and circular plasmids (30,45,47,48). The RAD1 function is also required for elevated recombination in cdc9 and top3 mutants (2, 47) as well as for mitotic recombination stimulated by RNA polymerase I-or II-dependent transcription (59, 67). The effect of rad1 and rad10 mutations on recombination seems to be rather specific in the sense that mitotic, but not meiotic, events are affected in the mutant strains (48). Moreover, only rad1 and rad10, not other NER mutants, showed defects in spontaneous recombination between directly repeated genes (47).A key to understanding a precise role of RAD1 in mitotic recombination came from our previous studies on the genetic control ...

DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

“…The counterpart of ERCC1-XPF in Saccharomyces cerevisiae, Rad10-Rad1, is required not only for NER but also for recombination between direct repeated DNA sequences, a process which is also called single strand annealing (13,14). By analogy, ERCC1-XPF is expected to fulfil a similar role in mammalian cells, and the two homologous protein complexes have been proposed to remove 3Ј protruding single-stranded arms from recombined duplex regions (13,15).…”

“…Incisions made by ERCC1-XPF and XPG during NER co-localize to the borders of the opened DNA intermediate (8) and protein-protein interactions with other NER factors are likely to determine the exact positioning of both nucleases. For ERCC1-XPF, interactions with XPA and RPA have been reported (9 -12).The counterpart of ERCC1-XPF in Saccharomyces cerevisiae, Rad10-Rad1, is required not only for NER but also for recombination between direct repeated DNA sequences, a process which is also called single strand annealing (13,14). By analogy, ERCC1-XPF is expected to fulfil a similar role in mammalian cells, and the two homologous protein complexes have been proposed to remove 3Ј protruding single-stranded arms from recombined duplex regions (13,15).…”

Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

“…These residual crossovers may reflect the activity of a yet-unidentified JM resolvase; they may also reflect the production of half-crossovers by breakinduced replication (Ho et al, 2010;Kogoma, 1996;Llorente et al, 2008) or by other mechanisms that do not involve dHJ-JM formation and resolution (Ivanov and Haber, 1995;Mazó n et al, 2012;Muñoz-Galván et al, 2012). Alternatively, long-tract NCO gene conversion events that include flanking heterologous sequences might be responsible for the products, scored in our molecular assays as COs, that are independent of both MutLg and SSNs.…”

Sources and Structures of Mitotic Crossovers That Arise When BLM Helicase Is Absent inDrosophila

“…Crossovers were significantly elevated in mei-9; Blm mutants relative to Blm single mutants, but were not elevated in mus201; Blm mutants ( Figure 1C). Rad1 has NER-independent DNA repair functions (Klein 1988;Fishman-Lobell and Haber 1992;Ivanov and Haber 1995); the crossover elevation caused by removing MEI-9 might be a consequence of disrupting pathways other than NER. We also tested the effect of removing the NER damage recognition protein XPC, which is encoded by the mus210 gene (Sekelsky et al 2000).…”