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dc.contributor.advisorPaull, Tanya T.
dc.creatorZhou, Yi, Ph. D.
dc.date.accessioned2018-03-19T17:12:34Z
dc.date.available2018-03-19T17:12:34Z
dc.date.created2015-12
dc.date.issued2015-12-09
dc.date.submittedDecember 2015
dc.identifierdoi:10.15781/T2GQ6RJ9J
dc.identifier.urihttp://hdl.handle.net/2152/63857
dc.description.abstractThe 5’ strand resection of DNA double strand breaks (DSBs) initiates homologous recombination (HR) and is critical for genomic stability. To date there is no quantitative method to measure single-stranded DNA (ssDNA) intermediates of resection in mammalian cells. In this study I develop a quantitative PCR (qPCR)-based assay to quantitate ssDNA intermediates, specifically, the 3’ ssDNA product of resection at specific DSBs induced by AsiSI restriction enzyme in human cells. I protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction, and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing protein foci-based methods. Using this assay I quantitatively measure ssDNA intermediates of resection in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle dependent manner. Depletion of CtIP, Mre11, Exo1, or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit (DNA-PKcs) leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of BRCA1 does not, suggesting that the role of BRCA1 in HR is primarily to promote RAD51 filament formation, not to regulate end resection. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, I also show that DNA-PKcs, a classic non-homologous end joining (NHEJ) factor, antagonizes DSB resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. ATM kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11/Rad50/Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation. This work suggests that, in addition to its key role in NHEJ, DNA-PKcs also acts in concert with MRN and ATM to regulate resection and thus DNA repair pathway choice. In addition, I find that MRN strongly suppresses DNA Ligase IV/XRCC4-mediated end rejoining, whereas it dramatically promotes DNA end ligation by the DNA Ligase III/XRCC1 complex. The Ataxia-Telangiectasia mutated (ATM) protein is a key regulator of checkpoint activation and HR in response to DSBs. The MRN complex acts as a DSB sensor and is essential for ATM recruitment to broken DNA ends and ATM activation. However, the precise mechanism for ATM activation upon DNA damage and ATM inactivation after DNA repair has remained poorly understood. Phosphorylation of ATM has been suggested to play important roles in this process. Autophosphorylation of ATM at four sites (S1981, S367, S1893, and S2996) has been shown to be essential for ATM activation and function in response to DNA damage in human cells. However, mutations at these four sites do not affect ATM kinase activity in vitro or in mouse models, suggesting that there are other mechanisms for regulation of ATM activity. Previous studies show that CDK5-mediated phosphorylation of ATM at Ser794 and EGFR-mediated phosphorylation of ATM at Tyr370 both positively regulate ATM activation upon DNA damage. In this study, I further propose that DNA-dependent protein kinase (DNA-PK) negatively regulates ATM activity through phosphorylation of ATM at multiple sites. ATM is hyperactive when DNA-PK activity is blocked by DNA-PK specific inhibitor or when the DNA-PKcs gene is deleted in cells. Pre-incubation of ATM protein with DNA-PK significantly inhibits ATM kinase activity in vitro. Using mass spectrometry analysis and site-directed mutagenesis, I characterized three clusters of sites: S85/T86, T372/T373 and T1985/S1987/S1988. The phospho-mimetic mutations at these residues repress ATM activation both in vitro and in cells. Overexpression of phospho-mimetic ATM mutants in ATM-deficient cells fails to restore cell survival, DSB end resection and G2/M checkpoint activation in response to DNA damage. In addition, I have observed that the phospho-blocking ATM mutants, T1985A/S1987A/S1988A and T86A/T373A, are resistant to DNA-PK pre-incubation in vitro and that overexpression of ATM T1985A/S1987A/S1988A mutant fails to respond to DNA-PK inhibition in comparison to wild-type ATM in cells. Taken together, my data suggests that the NHEJ factor DNA-PK suppresses the catalytic activity of ATM through phosphorylation and that the phosphorylation of a group of Ser/Thr residues on ATM negatively regulates ATM signaling upon DNA damage. Since ATM is known to promote the HR pathway, this may provide a novel mechanism for DNA repair pathway choice upon DNA damage as well as ATM inactivation after DNA repair.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.subjectDNA repair
dc.subjectDNA damage response
dc.subjectDouble strand breaks
dc.subjectATM
dc.subjectDNA-PKcs
dc.subjectMRN
dc.subjectExo1
dc.subjectDNA end resection
dc.titleRegulation of DNA damage response by ATM and DNA-PKcs
dc.typeThesis
dc.date.updated2018-03-19T17:12:34Z
dc.contributor.committeeMemberFinkelstein, Ilya J
dc.contributor.committeeMemberIyer, Vishwanath R
dc.contributor.committeeMemberMiller, Kyle M
dc.contributor.committeeMemberLee, Seongmin
dc.description.departmentCellular and Molecular Biology
thesis.degree.departmentCellular and Molecular Biology
thesis.degree.disciplineCell and Molecular Biology
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy
dc.type.materialtext


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