Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection, End-Tethering and Repair of a Double-Strand Break

Abstract

The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.

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

“…Further characterization of mre11-P110L showed that it is unable to suppress the subtle resection defect of the sae2Δ mutant, and that suppression of the sae2Δ CPT sensitivity is independent of the Mre11 nuclease activity and mostly Exo1 independent. By contrast, yku70Δ restores resection and resistance to DSBinducing agents to the sae2Δ mutant in an Exo1-dependent manner, and rad9Δ suppresses the sae2Δ end resection defect by allowing access to Sgs1-Dna2 (15,23,(38)(39)(40)(41). Deletion of DNL4 (encoding DNA ligase IV) does not suppress the DNA damage sensitivity of sae2Δ, mre11-3, and cpt1Δ mutants, indicating that the yku70Δ suppression is by allowing Exo1 access to ends and not by diverting ends from NHEJ to HR (23,28,38).…”

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…In the absence of Sae2, DSB repair is impaired due to prolonged MRX complex binding. This defect is rescued by depleting the yeast 53BP1-ortholog RAD9, as 53BP1 promotes NHEJ by inhibiting dsDNA resection for HR (106, 107). In humans, the tumor suppressor BRCA1 inhibits 53BP1 at breaks in association with MRN and CtIP to promote HR over NHEJ (107–110).…”

Mechanism and regulation of DNA end resection in eukaryotes

“…Rad9 also prevents extensive resection by Exo1 and Dna2-STR at uncapped telomeres (Ngo et al, 2014; Ngo and Lydall, 2010). 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). The mechanism by which Rad9 inhibits resection is not fully understood, but is dependent on chromatin association and oligomerization of Rad9 suggesting it forms a physical barrier to Exo1 and Dna2-STR (Ferrari et al, 2015). Activation of Rad53 could also contribute to the Rad9 inhibition of end resection (see below).…”