Roles of cancer, aging, cell cycle and telomere associated proteins in mammalian DNA double strand break repair

DNA double strand breaks (DSBs) are dangerous lesions that may lead to massive loss of genetic information and cell death. Cells have evolved sophisticated machinery to repair DNA DSBs. There are two major pathways of DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Although the major enzymes involved in NHEJ and HR had been identified the interaction between these two pathways and their regulation is poorly understood.;The goal of my thesis has been to study the mechanisms of NHEJ and HR; to understand the relationship between these two pathways; and to examine the changes in DSB repair during aging and tumorigenesis.;To facilitate the analysis of DSB repair, I developed reporter cell lines for detecting the efficiency of NHEJ and HR in vivo. Using this system, I first examined the relative contributions of NHEJ and HR to DNA DSB repair and the kinetics of the DSB repair events. We found that in normal human cells NHEJ is responsible for 75% of repaired DNA DSBs while HR repairs the remaining 25%. In addition, we demonstrated that NHEJ is a fast process, which takes approximately half an hour to complete, while HR takes at least 7 hours. Next, I analyzed the efficiency of NHEJ and HR at different cell cycle stages in normal human cells with intact cell cycle checkpoints. I showed that NHEJ is active throughout the cell cycle but its efficiency is higher in the S and G2/M phases while HR functions predominantly in the S phase.;I demonstrated that TRF2, an essential telomeric binding factor, is required for repair of DNA DSBs by HR. Depletion of TRF2 dramatically inhibited HR and slowed down the recruitment of Rad51 to the broken ends, suggesting that TRF2 is involved in forming and stabilizing Holliday Junctions.;I systematically analyzed the efficiency of NHEJ and HR in a collection of breast cancer cell lines and normal mammary epithelial cells. I discovered that HR is elevated in breast cancer cells compared to normal breast epithelial cells. In addition, I found that breast cancer cells were more sensitive to irradiation than normal epithelial cells.;Using the reporter cassette for measuring HR in normal human fibroblast HCA2, I found that the efficiency of HR declines as the cells progress towards senescence. The decrease was caused by an onset of irreversible cell cycle arrest and a reduction in expression of HR proteins.;I found that SIRT6, a mammalian homolog of the yeast Sir2 protein, is required for DNA DSB repair by both NHEJ and HR under oxidative stress. I further demonstrated that SIRT6 interacts with PARP1 and promotes mono-ADP-ribosylation of PARP1, thereby stimulating the poly-ADP-ribosylase activity of PARP1. Activation of PARP1 by SIRT6 leads to a strong increase in efficiency of NHEJ and HR under stress. I propose that SIRT6 functions as a regulator integrating oxidative stress signaling and DNA damage response.