Sae2 promotes DNA damage resistance by removing the Mre11–Rad50–Xrs2 complex from DNA and attenuating Rad53 signaling

Abstract

The Mre11-Rad50-Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA doublestrand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpinopening defects of the sae2Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M P110L RX complex shows reduced binding to single-and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2Δ cells to clastogens.DNA repair | Mre11 | Sae2 | DNA damage checkpoint

DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination

“…Thus, in addition to stimulating the Mre11 endonuclease, Sae2 has other, Mre11-nuclease independent roles. This may include its proposed function to remove MRX from DNA ends upon end processing to facilitate downstream repair, attenuate checkpoint signaling, counteract the NHEJ factor Ku, and promote resection by Exo1 (26,29,(57)(58)(59). Sae2 itself was also shown to possess a nuclease activity specific to secondary structures in DNA (60), although an enzymatic activity was not detected by other laboratories (27,50).…”

When two is not enough: a CtIP tetramer is required for DNA repair by Homologous Recombination

“…8,9 The critical function of CtIP/Ctp1/Sae2 at DNA ends is carried out in close functional and physical cooperation with the DNA damage sensor and repair complex MRE11-RAD50-NBS1 (MRN, where NBS1 is Xrs2 in budding yeast). [10][11][12][13][14][15] The central role of CtIP/Ctp1/Sae2 in DSB repair is further highlighted by its complex regulation, mediated by an array of post-translational modifications that include phosphorylation by cyclin-dependent and DNA-damage activated kinases, acetylation, ubiquitylation, NEDDylation and proline isomerisation. 3 Despite its importance to DSB repair, our mechanistic understanding of how CtIP promotes resection remains incomplete.…”

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae

“…SAE2 is required for activating Mre11 endonuclease activity (Cannavo and Cejka 2014). Given the strong genetic interaction with sae2D, we hypothesized that Thr675 on Xrs2 may function in a parallel pathway of activating Mre11 endonuclease activity, which is essential for timely removal of the MRX complex from DSB ends, and the retention of MRX results in prolonged checkpoint activation and growth defects (Clerici et al 2006;Bernstein et al 2013;Chen et al 2015). Our genetic studies showed that tel1D and mec1D checkpoint mutants suppressed the severe sensitivity of an xrs2 S349A, T675A sae2D strain to MMS ( Figure 5B), consistent with the idea that prolonged checkpoint activation is responsible for the severe MMS-sensitivity phenotype.…”

“…The Mre11 endonuclease activity (promoted by Sae2) is known to be required for removing the MRX complex from DSB ends to facilitate subsequent long-range resection (Clerici et al 2006;Bernstein et al 2013;Chen et al 2015). Retention of MRX on the damage site results in prolonged checkpoint activation and growth defects (Clerici et al 2006;Chen et al 2015).…”

“…Retention of MRX on the damage site results in prolonged checkpoint activation and growth defects (Clerici et al 2006;Chen et al 2015). One hypothesis to explain the strong interaction between xrs2 S349A, T675A and sae2D is that xrs2 S349A, T675A may also be defective in activating the endonuclease activity of Mre11 (without disruption of MRX); thus mutation of Ser349 and Thr675 on Xrs2 coupled with loss of Sae2 may cause drastic inactivation of Mre11 endonuclease activity, leading to retention of MRX and uncontrolled checkpoint activation.…”