Interplay of Ku antigen and poly(ADP-ribose) polymerase-1 in the regulation of non homologous DNA-end joining

Title: Interplay of Ku antigen and poly(ADP-ribose) polymerase-1 in the regulation of non homologous DNA-end joining
Authors: Crawley, Clayton D
Date: 2009
Abstract: The detection and repair of double-stranded DNA breaks (DSBs) is crucial to the functioning of the cell and the integrity of the genome. In higher eukaryotes, the non-homologous end joining (NHEJ) pathway is the more widely used mechanism for repairing DSBs. The NHEJ pathway is initiated by the binding of Ku to the DSB. Ku is a ring-shaped heterodimer that binds to DNA with high affinity by threading the DNA through its central pore. Current models of the NHEJ pathway suggest that as repair proceeds, Ku is pushed to the interior of the DNA, away from the ends. According to these scenarios, upon repair of the break Ku would be trapped on the ligated DNA without a means of dissociating from the DNA once repair is completed. PARP1 is member of the poly(ADP-ribose) polymerase (PARP) enzyme family, and is believed to be involved in the resolution of DSBs. When active, PARP1 transfers and polymerizes ADP-ribose polymers to target nuclear proteins, which in turn, alters their activity. The enzymatic activity of PARP1 is stimulated by the binding of PARP1 to DNA breaks and strand interruptions. Recent work has suggested a functional interaction between Ku and PARP1 in DNA repair. Given reports that PARP1 can affect the affinity of Ku for DNA and the enzymatic role of PARP1, I have developed the hypothesis that PARP1 stimulates the release of Ku from DNA. My work in this thesis has shown that the release of Ku from DNA is dramatically accelerated by the addition of PARP1 under ADP-ribosylation permissive conditions. This release is dependent upon the enzymatic activity of PARP1 rather than the presence of PAR polymers. The PARP-mediated release of Ku from DNA is not dependent upon the presence of free ends as Ku was no longer detected on closed circular DNA after incubation with PARP1. PARP1 ADP-ribosylates three glutamate residues within the C-terminal SAP domain of the Ku70 subunit of the heterodimer, and mutation of these residues abrogates the effect of PARP1 on Ku. The ribosylation of Ku by PARP1 is necessary for an appropriate DNA damage response in vivo, as mutation of these residues resulted in an increased sensitivity to DNA damaging agents and a prolonged retention of Ku at repair sites. In addition to the deficiencies in responding to DNA damage the mutations resulted in dramatically reduced V(D)J recombination potential and defects in the formation of signal joints. The findings from the current study provide a novel and significant insight into the importance of ribosylation in regulating Ku function in DNA repair, and imply a further significance for ribosylation in regulating other roles of Ku.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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