BRCA1Modulates The Autophosphorylation Status Of DNA-PKcs In S Phase Of Cell Cycle

DNA double-strand break (DSB) can be induced by a variety of stimuli from endogenous sources such as reactive oxygen species (ROS) generated during cellular metabolism, replication stress, and V(D)J recombination, or from exogenous sources such as ionizing radiation (IR) and chemotherapeutic agents. Unrepaired or misrepaired DSB can result in senescence, chromosomal aberrations, including translocations and deletions, or cell death. The chromosomal aberrations can lead to genomic instability and tumorigenesis. To counteract the effects of DSB, two highly efficient DSB repair pathways have been evolved in eukaryotic cells:non-homologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ pathway repairs DSB via direct ligation of the broken DNA molecule; whereas HR utilizes an unbroken, homologous DNA template for replication. NHEJ is initiated by the association of the Ku70/80heterodimer to DNA ends. Ku70/80then recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to DSB ends. DNA-PKcs along with Ku70/80forms a distinct structure at the DNA termini, which is likely to play an active role in the formation of a synaptic complex that holds the two ends of the broken DNA molecule together. After end processing, the two tethered DNA termini are ligated. On the other hand, HR is initiated once the5’-3’resection of the DSB occurs. The5’-3’resection creates single strand DNA (ssDNA) ends which are used for strand invasion and exchange into a homologous DNA template. Following DNA synthesis, ligation, and branch migration, the recombination intermediates (Holliday junctions) are resolved and the break is completely repaired. Although, much work has been performed to identify and characterize factors which are required for both DSB repair pathways, many important questions remain unresolved, including which factors initially bind to the DSB ends and stabilizes them and what is the mechanism that modulates the pathway choice between NHEJ and HR for the repair of DSB.NHEJ is not restricted to any cell cycle stage, whereas HR is active in the S/G2phases. The difference in cell cycle specificity between these two DSB repair pathways suggests there are cell cycle specific mechanisms for the pathway choice between NHEJ and HR. In the present study, we show that NHEJ is attenuated in S phase of the cell cycle via modulating autophosphorylation of DNA-PKcs at serine2056. First, using live cell imaging and micro-laser system, we found that the NHEJ factor DNA-PKcs can be recruited to laser-generated DSB and it’s initial association and kinetics was similar in S phase and non-S phase cells, suggesting that DNA-PKcs can be recruit to DSB in any phase of cell cycle. Next, the BRCA1was found to be responsible for the attenuation of DNA-PKcs autophosphorylation at serine2056in S phase. In the BRCA1defective cell line HCC1937, autophosphorylation of DNA-PKcs S2056was similar in S phase and non-S phase cells. While in the HCC1937cells complemented with BRCA1, autophosphorylation of DNA-PKcs S2056was markedly reduced in S phase cells compared with non-S phase cells. We found that BRCA1interacts with DNA-PKcs in a cell cycle-dependent manner and this interaction is not induced by DNA damange. Also, this interaction is phosphorylation-independent. Futher-more, the tandem BRCT domains of BRCA1was found to interact with N terminal region of DNA-PKcs around S2056cluster. This interaction directly blocks the ability of DNA-PKcs S2056to autophosphorylate in vitro and in vivo and moderately affects its overall kinase activity. We further found that BRCA1did not affect the ability of DNA-PKcs to localize to or its association dynamics at DSB. Finally, blocking autophosphorylation of DNA-PKcs results in a increase in HR-mediated repair as monitored by significant increases in DNA end resection and focus formation of the HR factor Rad51after IR. In summary, BRCA1regulates DSB repair pathway choice by modulating autophosphorylation of DNA-PKcs.