Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity

Abstract

The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery.

Xrs2 Dependent and Independent Functions of the Mre11-Rad50 Complex

“…While the enrichment of Mre11 at the DSB was comparable in MRE11 and MRE11-NLS cells, less Mre11-NLS was retained at the DSB in the xrs2Δ background at the 1 hr time point ( P <0.05) (Fig 3C). Since Tel1 signaling is abrogated in the xrs2Δ MRE11-NLS mutant, and Tel1 is required for retention of Mre11 at DSBs (Gobbini et al, 2015), we considered the possibility that the Mre11 localization defect in xrs2Δ MRE11-NLS cells could be due to loss of Tel1 recruitment. Indeed, we found that Mre11 enrichment at the HO-induced DSB was the same in MRE11-NLS xrs2Δ and MRE11-NLS cells tel1Δ (Fig 3C).…”

“…Interestingly, we found the level of Dna2 enrichment 1 kb from the DSB was not significantly decreased in the MRE11-NLS xrs2Δ strain, despite the decrease in Mre11, whereas Dna2 enrichment was barely above background in MRE11-NLS sae2Δ xrs2Δ cells (Fig 3C). The decreased Dna2 binding in the MRE11-NLS sae2Δ mutant could be due to delayed resection initiation or Rad9 inhibition (Ferrari et al, 2015; Gobbini et al, 2015). We suggest that there are normally two modes of Sgs1-Dna2 recruitment to DSBs: via MRX interaction and by RPA interaction after MRX-Sae2 dependent cleavage creates ssDNA (Fig 3D) (Chen et al, 2013; Shim et al, 2010).…”

“…Previous studies have shown Xrs2/Nbs1 binds to DNA, and to the Mre11 latching loop, potentially stabilizing the Mre11 dimer at ends (Paull and Gellert, 1999; Schiller et al, 2012; Trujillo et al, 2003). Mre11 retention was equally reduced in the MRE11-NLS xrs2Δ and MRE11-NLS tel1Δ mutants suggesting the xrs2Δ defect could result from failure to recruit Tel1 (Gobbini et al, 2015). However, it is important to note that tel1Δ mutants are proficient for end resection, hairpin resolution and meiosis indicating that the reduction in Mre11 retention at DNA ends is of no consequence for these Mre11-dependent functions (Carballo et al, 2008; Iwasaki et al, 2016): thus, Xrs2 must contribute in some way to Mre11 activity, independent of the role of Tel1 stabilization of Mre11 at ends.…”

Mechanism and regulation of DNA end resection in eukaryotes

“…Elimination of Rad9 can suppress the sae2Δ resection initiation defect by allowing more efficient recruitment of Dna2-STR to DSBs (Bonetti et al, 2015; Ferrari et al, 2015). Similarly, partial loss of function alleles of TEL1 and RAD53 that reduce Rad9 accumulation at DSBs can also suppress the sae2Δ resection defect (Gobbini et al, 2015). Elimination of Tel1 causes a modest delay in resection initiation while the mec1Δ mutant exhibits increased end resection due to reduced Rad9 recruitment (Clerici et al, 2014; Mantiero et al, 2007).…”

DNA damage kinase signaling: checkpoint and repair at 30 years

“…Such stabilization of Slx4 at DNA lesions, which is also dependent on Rtt107's interaction with phosphorylated H2A (Fig ), is believed to counteract a resection block promoted by Rad9, thereby allowing long‐range resection to occur (Dibitetto et al , ; Liu et al , ). Importantly, the role of Rad9 in counteracting resection relies on its oligomerization and Rad53 signaling (Clerici et al , ; Ferrari et al , ; Gobbini et al , ), which are both dependent on Mec1‐mediated phosphorylation events. Therefore, Mec1 plays opposing roles in HR, inhibiting resection via the Rad9–Rad53 signaling axis or promoting resection by mediating the Slx4–Dpb11 interaction (Fig ).…”