Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination.

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.

Shao Z ，Davis AJ ，Fattah KR ，So S ，Sun J ，Lee KJ ，Harrison L ，Yang J ，Chen DJ ... -  《-》

Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

Lee KJ ，Saha J ，Sun J ，Fattah KR ，Wang SC ，Jakob B ，Chi L ，Wang SY ，Taucher-Scholz G ，Davis AJ ，Chen DJ ... -  《-》

Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier.

Willis NA ，Panday A ，Duffey EE ，Scully R ... -  《PLoS Genetics》

Chloroethylnitrosourea-induced cell death and genotoxicity: cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ.

Nikolova T ，Hennekes F ，Bhatti A ，Kaina B ... -  《-》

Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

Castore R ，Hughes C ，Debeaux A ，Sun J ，Zeng C ，Wang SY ，Tatchell K ，Shi R ，Lee KJ ，Chen DJ ，Harrison L ... -  《-》