Effects Of Ku70/80Binding At DNA Double Strands Break Ends On Homologous Recombination

Our genetic material DNA is continually threatened by exogenous damage factors like chemicals, ionizing radiation (IR) and ultraviolet, as well as endogenous damage factors including metabolic products and reactive oxygen species (ROS). DNA double strands break (DSB) is one of the most harmful kinds of DNA damage as unrepaired or misrepaired DSBs can lead to genome rearrangement, apoptosis, tumorigenesis and immune deficiency. Eukaryotic cells protect genome integrity by DNA damage response (DDR) network, which initiates a series of signal transduction cascades to subsequently lead to DNA repair, apoptosis, chromatin remodeling, etc. Upon activation of DSB, DDR initiate repair by cell-cycle arrest and two DSB repair pathway:homologous recombination (HR) and nonhomologous end joining (NHEJ).HR requires homologous templates to conduct a high-fidelity repair; in contrast, NHEJ utilizes enzymes to directly ligate the two broken ends and is thus less reliable, but is capable of rejoining almost any kind of DSB ends. Different cell types have preference in the two pathway, in addition, two other key factors also participate in modulating DSB repair pathway thoice:cell cycle stages and DNA ends resection. HR prefers sister chromatins as homologous templates and is therefore assumed to be mainly active in S/G2phases when sister chromatins are accessible. NHEJ doesn't have restrictions to any specific cell cycle phases but is believed to be dominant in GO and G1phases. HR needs DNA ends resection to generate a3'overhang for strand invasion and pairing, and since NHEJ initiator Ku70/80has high affinity to dsDNA (double strands DNA) but less affinity to ssDNA (single strand DNA) so end resection pushes DSB repair to HR pathway. The direct competition model assumes that end resection factors and Ku70/80directly compete for the DSB ends in S/G2phase of the cell cycle, and end resection factors bind to DSB ends first, rapidly forming ssDNA to prevent Ku binding, thus HR becomes the dominant pathway. Direct competition model is supported by several evidences but was recently weakened by data showing that Ku70/80has higher affinity for DNA than the MRX complex. So we conducted this study to verify whether Ku70/80only binds to DSB ends in specific phases of cell cycle, and how this binding would affect HR.It has been shown here that although NHEJ pathway is thought to be dominant only in GO and G1phase,-Ku80was also recruited to365nm pulsed nitrogen laser caused DSB sites in S phase mammalian cells when HR is assumed to be dominant. Moreover, It's initial association and dynamics/kinetics at DSBs was similar to non-S phase mammalian cells. A Ku homolog from Mycobacterium tuberculosis (Mt-Ku) was found to be localized to and be retained at DSB sites after laser generated DNA damage in S phase, and was thus used to determine whether sustained blocking of DSB ends would affect end resection and HR pathway in mammalian cells. The3'overhang ssDNA generated by end resection is immediately coated by RPA; and BrdU antibody only recognizes BrdU integrated in ssDNA, so RPA and BrdU foci formation were ultilized as indirect and direct measurement of ssDNA formation following DNA damage. Rad51is an indispensable HR factor mediating end invasion and pairing, and is thus a well established marker for HR mediated repair. We observed here that when Ku deficient rodent cells were complemented with Mt-Ku, a marked decrease in RPA, 3rdU, and Rad51foci formation occurred after ionizing radiation, suggesting a decrease n DNA end resection and HR mediated repair. Further confirming of this, HR assay ilso showed less percentage of HR repair accomplished cells in Mt-Ku complemented cells compared to uncomplemented cells. And Mt-Ku complementation resuled in an even higher IR sensitivity implying that this NHEJ non-functioning Ku also inhibited HR by its persistently binding to the DSB ends.We further investigated the possible mechanism mediating human Ku70/80dissociation from DSB sites. Previous results have shown that treatment with phosphatidylinositol3-kinase-like protein kinase (PIKK) inhibitor wortmannin could retain Ku80at DSB ends upto120min after laser damage in human cells. In addition, we've shown here that wortmannin also induced decreased RPA and Rad51foci formation after IR, revealing a role of Ku phosphorylation status in it dissociation. To identify the specific kinase mediating Ku dissociation related phosphorylation, we examined the function of ATM specific inhibitor KU-55933and DNA-PKcs specific inhibitor NU-7441. NU-7441successfully induced sustained Ku80binding at DSB sites, and partially reduced RPA formation after IR. Alghough KU-55933alone did not show significant effect on Ku80kinetics, when combined with NU-7441, together they showed significantly enhanced DSB end blocking effect by causing even severe reduction of RPA foci post-IR. These results indicated that dissociation of Ku from DSB ends is related toDNA-PKcs mediated phosphorylation and ATM could enhance such effect of DNA-PKcs. Taken together, all the above data suggest that Ku70/80binds to DSBs regardless of cell cycle phases and may be actively displaced from DSB ends via phosphorylation by DNA-PKcs, probably with the help from ATM, to free the DNA ends for DNA end resection and HR to occur.