Abstract
Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.
Highlights
Repair of single-ended DNA double-strand breaks by homologous recombination (HR) requires the generation of a 30 single-strand DNA overhang by exonuclease activities in a process called DNA resection
Because DNA-PK activity requires Ku binding to double-strand break (DSB), these findings implied that an active DNA-PK complex, composed of the Ku70-Ku80 dimer and DNA-PKcs, assembles on and is activated by single-ended DSBs (seDSBs) generated by CPT
By using high-resolution imaging, we examined individual seDSBs marked by resection-dependent RPA70 foci and directed towards HR-repair
Summary
Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 30 single-strand DNA overhang by exonuclease activities in a process called DNA resection. The discovery of MRE11 endo- and exonuclease inhibitors has recently supported the existence of a similar mechanism in higher organisms[18,19] This mode of ‘bi-directional resection’ helps explain how resection can be initiated in the presence of Ku, but still leaves the issue of how Ku is removed from DNA ends to allow HR to proceed. We establish that ATM-dependent phosphorylation of CtIP plus MRE11 endonuclease activity counteract Ku accumulation at seDSBs. we show that downstream of MRE11 endonuclease activity, the epistatic action of MRE11 exonuclease activity and the recently discovered CtIP flap endonuclease activity[24,25] are required to antagonize Ku, and for efficient RAD51 loading at seDSBs. Our work provides evidence for a hitherto unsuspected mechanism operating in parallel to MRE11 exonuclease and CtIP flapendonuclease activities to counteract Ku persisting at seDSBs. We propose that MRE11 endonuclease and exonuclease activities process the DNA flanking Ku, and prime Ku for release by CtIP 50-flap endonuclease activity. We anticipate that ‘attacking DNA ends from the flanks’ through the coordinated action of CtIP and MRE11 nuclease activities is a general mechanism to repair complex DNA lesions congested with proteins or bulky DNA adducts
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
