Local unwinding of double-strand DNA ends by the MRX complex promotes Exo1 processing activity

Abstract

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs), which involves different nucleases including the Mre11-Rad50-Xrs2 (MRX) complex and the Exonuclease 1 (Exo1). The characterization of a novel mutation in Mre11 causing accelerated DSB resection has allowed to show that MRX facilitates DNA end processing by Exo1 through local unwinding of double-stranded DNA ends.

MRE11-RAD50-NBS1 Complex Is Sufficient to Promote Transcription by RNA Polymerase II at Double-Strand Breaks by Melting DNA Ends

“…Importantly, the MRN complex also has the capacity to transiently separate individual DNA strands of a duplex, although its relevance for DSB repair remains to be fully understood ( Cannon et al., 2013 ; Gobbini et al., 2018 ; Liu et al., 2016 ; Nicolette et al., 2010 ; Williams et al., 2008 ). Specifically, it is not clear whether dsDNA melting is a prerequisite for the clipping of blocked DNA ends by MRN-CtIP.…”

“…DNA melting by MRN is stimulated by ATP binding and hydrolysis by RAD50, and this is important for MRN binding to DNA ( Liu et al., 2016 ; ( Lee and Paull, 2004 ); Paull, 2015 ). The DNA melting activity of yeast homolog MRX has been shown to enhance the resection activity of ExoI in Saccharomyces cerevisiae ( Gobbini et al., 2018 ).…”

Mapping yeast mitotic 5’ resection at base resolution reveals the sequence and positional dependence of nucleasesin vivo

“…To add information to the signal patterns above we repeated resection sequencing in strains lacking Exo1 or Sgs1, which are required for the two main long-range resection mechanisms (Mimitou and Symington, 2008;Zhu et al, 2008). While the sgs1Δ resection profile looked largely like that of WT (Figure 2A), exo1Δ first led to a decrease in reads aligning near the HOcs, especially at the -2 position, consistent with Exo1 itself being responsible for removing a limited number of terminal bases (Figure 2A-B) (Gobbini et al, 2018). In marked contrast, we observed a dramatic increase in reads mapping to the -65/-66 bp position in the absence of Exo1 (Figure 2A,C), indicating that Exo1 did not create this signal but instead was responsible for suppressing it in WT cells.…”

“…Notably, signal at the -2 position was uniquely dependent on Exo1, which identifies Exo1 as the nuclease that cleaves some, but not all, of the DSB-proximal 5' intermediates (Gobbini et al, 2018). In contrast to Ku and Nej1, loss of Mre11 or its nuclease activity led to increased signal at the DSB-proximal positions, most profoundly at -2 bp, with a similar result upon loss of the Tel1 and Sae2 proteins that support Mre11 (Cannavo and Cejka, 2014;Cassani et al, 2019;Yu et al, 2019).…”

“…These are not exclusive concepts and the fact that signal at the prominent −2 position failed to accumulate in Ku mutant yeast suggests that NHEJ factors might normally but only temporarily stabilize DSB-proximal intermediates. Notably, signal at the −2 position was uniquely dependent on Exo1, which identifies Exo1 as the nuclease that cleaves to some, but not all, of the DSB-proximal 5’ intermediates (39). In contrast to Ku and Nej1, loss of Mre11 or its nuclease activity led to increased signal at the DSB-proximal positions, most profoundly at −2 bp, with a similar result upon loss of the Tel1 and Sae2 proteins that support Mre11 (10, 16, 40).…”