Abstract
DNA-Pk is the essential component of nonhomologous end joining (NHEJ) pathway for DNA-double strand break (DSB) repair. While NHEJ is predominately active in G1, it is not clear how interplay between NHEJ and homologous recombination (HR) in S or G2 phases of the cell cycle. We have tested the hypotheses that DNA-Pk plays an active role in modulating the interplay between NHEJ and HR in S phase of the cell cycle. I'll first discuss the role of Ku in modulating DNA end resection; subsequently, I'll discuss a potential role of phosphorylation status of DNA-PKcs at T2609 cluster in modulating HR. We have studied the inhibition of DNA end resection in vitro mediated by human Exo1 in the presence of Ku70/80, DNA-PKcs, Mre11 or MRN complex using purified proteins. Our results showed that Ku blocks DNA end resection (5'-3') mediated by Exo1 if Ku is bound to dsDNA. Such inhibition cannot be reversed by the addition of Mre11 or MRN complex. Further more, when DSB ends are persistently occupied by Ku, DNA end resection is blocked and subsequently HR is attenuated. In addition, when DNA-PKcs phosphorylation at T2609 cluster is blocked, it affects both NHEJ and HR. Hamster cells with T2609 S/T to A mutations (6A) are highly sensitive to MMC, CPT and UV in addition to IR. Further more, 6A mutant is sensitive to IR in S phase of the cell cycle compares with the null mutation and defective in I-sceI mediated HR. Taken together, our results support the hypothesis that Ku is recruited to DSBs, stabilizing the ends and interplay between HR and NHEJ in S phase of the cell cycle. In addition, phosphorylation status of DNA-PKcs at T2609 clusters further modulating the HR pathway of DSB repair.
