Abstract
Replication-associated recombinational repair is important for genome duplication and cell survival under DNA damage conditions. Several nonclassical recombination factors have been implicated in this process, but their functional relationships are not clear. Here, we show that three of these factors, Mph1, Mms2, and the Shu complex, can act independently to promote the formation of recombination intermediates during impaired replication. However, their functions become detrimental when cells lack the Smc5/6 complex or Esc2. We show that mph1Delta, mms2Delta, and shu1Delta suppress the sensitivity to the replication-blocking agent methylmethane sulfonate (MMS) in smc6 mutants, with double deletions conferring stronger suppression. These deletion mutations also rescue the MMS sensitivity of esc2Delta cells. In addition, two-dimensional gel analysis demonstrates that mph1Delta, mms2Delta, and shu1Delta each reduce the level of recombination intermediates in an smc6 mutant when cells replicate in the presence of MMS, and that double deletions lead to a greater reduction. Our work thus suggests that Mph1, Mms2, and the Shu complex can function in distinct pathways in replication-associated recombinational repair and that the Smc5/6 complex and Esc2 prevent the accumulation of toxic recombination intermediates generated in these processes.
Highlights
Recombinational repair provides an important means to facilitate replication when DNA lesions or other obstacles are present on the template
Our results suggest that Mph1, the Shu complex, and Mms2 can act independently to promote the formation of recombination structures, and that the Smc5/6 complex and Esc2 prevent toxicity from unresolved recombination intermediates generated by Mph1, Shu complex, and Mms2-dependent processes
Previous studies have shown that mutants of Mph1 and the Shu complex display moderate methylmethane sulfonate (MMS) sensitivity and have epistatic relationships with rad51⌬ (Schurer et al, 2004; Shor et al, 2005; Mankouri et al, 2007; Chen et al, 2009)
Summary
Recombinational repair provides an important means to facilitate replication when DNA lesions or other obstacles are present on the template. Several modes of replication-associated recombinational repair have been proposed These include gap filling that repairs single-stranded DNA regions left behind by the replication machinery, template switching that entails the use of newly synthesized sister strands as templates to overcome lesions on parental strands, and replication fork regression in which the newly synthesized DNA strand anneal to each other leading to DNA synthesis and/or strand invasion (Branzei and Foiani, 2007; Lambert et al, 2007; Li and Heyer, 2008; Budzowska and Kanaar, 2009; Chang and Cimprich, 2009). Some of these factors are not crucial for other recombination processes, such as the repair of double-strand breaks in a setting that is not coupled with replication
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