Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

Abstract

Repair of DNA bulky lesions often involves multiple repair pathways such as nucleotide-excision repair, translesion DNA synthesis (TLS), and homologous recombination (HR). Although there is considerable information about individual pathways, little is known about the complex interactions or extent to which damage in single strands, such as the damage generated by UV, can result in double-strand breaks (DSBs) and/or generate HR. We investigated the consequences of UV-induced lesions in nonreplicating G2 cells of budding yeast. In contrast to WT cells, there was a dramatic increase in ssDNA gaps for cells deficient in the TLS polymerases η (Rad30) and ζ (Rev3). Surprisingly, repair in TLS-deficient G2 cells required HR repair genes RAD51 and RAD52, directly revealing a redundancy of TLS and HR functions in repair of ssDNAs. Using a physical assay that detects recombination between circular sister chromatids within a few hours after UV, we show an approximate three-fold increase in recombinants in the TLS mutants over that in WT cells. The recombination, which required RAD51 and RAD52, does not appear to be caused by DSBs, because a dose of ionizing radiation producing 20 times more DSBs was much less efficient than UV in producing recombinants. Thus, in addition to revealing TLS and HR functional redundancy, we establish that UV-induced recombination in TLS mutants is not attributable to DSBs. These findings suggest that ssDNA that might originate during the repair of closely opposed lesions or of ssDNA-containing lesions or from uncoupled replication may drive recombination directly in various species, including humans.

Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

“…For example, γ-rays and UV induce mainly DSBs or single-stranded gaps (Ma et al 2013), respectively, while CPT leads to covalently bound topoisomerase I to the 3′ phosphate end at a nick (Pommier 2009). Replication of the unremoved adduct leads to a DSB in the next round of replication.…”

Slx4 scaffolding in homologous recombination and checkpoint control: lessons from yeast

“…While the role of resection in the repair of DSBs by HR is well established, less is known about the importance of resection for the repair of ssDNA gaps by HR. Evidence suggest that expansion of the ssDNA gaps by the action of exonucleases, especially Exo1, would be important to facilitate topological DNA transactions mediated by recombination factors (Giannattasio et al 2010; Karras et al 2013; Ma et al 2013). In agreement with that, Exo1 overexpression can partially rescue the MMS sensitivity of slx4Δ cells (unpublished observations).…”

Long repeating (TTAGGG) single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation

“…DNA replication, transcription, recombination and repair all involve the generation of single-stranded DNA (ssDNA) 4 seg-ments (1)(2)(3). In some cases, such as resection of DNA ends during repair or creation of an uncoupled replication fork, these ssDNA segments potentially could span several hundred nucleotides or more in length (4). Mixed sequence ssDNA segments are assumed to behave as random coils with some (weak) secondary structure caused by local base pairing (5,6).…”