Browsing by Subject "base excision repair"
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Item Protein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B(2003-12-01) Confer, Nils Forgard; Alvarez, Rafael; Ben S. Aar…; Wu, MingehiConfer, Nils Forgard, Protein-Protein Interactions Between Poly(ADP-ribose) Polymerase-1 and DNA Polymerase B. Doctor of Philosophy (Biomedical Sciences), December 2003, 114 Pages, 22 Figures, 1 Graph, and 80 References. The mammalian genome is continually subjected to chemical and environmental modifications that are repaired by base excision, and when excessive, may lead to apoptosis. Interestingly, the chromosomal enzyme poly(ADP-ribose) polymerase-1 (PARP-1) appears to modulate both mechanisms, either facilitating DNA repair and/or modulating cell death. In this dissertation project, experiments were performed to address the regulatory potential of PARP-1 in base excision repair (BER) and specifically on DNA polymerase B (pol B) function. Activity gels were used to measure the DNA polymerase activity of pol B following protein-(ADP-ribosyl)ation. However, the fraction of pol B molecules (ADP-ribosyl)ated was never 100% under the reaction conditions employed. In fact, similar results were observed in activity gels specific for PARP-1, even under conditions where this polymerase is the primary nuclear acceptor for poly(ADP-ribose) Here, I also describe a newly developed electrophoretic-mobility-shift-assay (EMSA) to monitor for the specific binding of pol B to a custom-made five-nucleotide gapped DNA duplex. However, while specific for pol B, this assay was inefficient to monitor the effects of covalent poly(ADP-ribosyl)ation on pol B activity. Moreover, I also observed the specific molecular association of PARP-1 is specifically proteolyzed into peptide fragments by caspases, conditions were established for the efficient proteolysis of PARP-1 by either capase-7. Experimental results indicated that caspase-3 was more efficient than caspase-7 at splitting unmodified PARP-1 into two peptide fragments. By contrast, caspase-7 appeared best suited for the proteolysis of covalently auto-poly(ADP-ribosyl)ated-(PARP-1). Interestingly, both of the caspase-generated peptide fragments of PARP-1 specifically associated with pol B as supported by co-immunoprecipitation/immune-blotting experiments. Taken together, the experimental results presented here support the hypothesis that a molecular mechanism exists that involves interaction(s) of PARP-1 with pol B that may help to facilitate the decision making process between cell survival and cell death. Thus, upon proteolytic degredation of PARP-1 into a 24-kDa amino-terminal fragment and an 89-kDa carboxy-terminus, each truncated peptide, separately, retains physical association with pol B, and inhibits DNA repair associated pol B activity to irreversibly switch the fate of cell from BER toward chromatin degradation and, eventually, programmed cell death.Item Studies of Checkpoint Responses Caused by Endogenous Oxidative DNA Damage in DNA Repair Deficient Saccharomyces Cerevisiae(2008-12-01) Pawar, Vaibhav; Alvarez, Rafael; Jiang, Yiwei; Krishnamoorthy, Raghu R.Pawar, Vaibhav., Studies of checkpoint responses caused by endogenous oxidative DNA damage in DNA repair deficient Saccharomyces cerevisiae. Doctor of Philosophy (Biomedical Sciences), December 2008, 163 pp., 21 illustrations, 189 references. In this dissertation project, I aimed to study checkpoint response of stationary phase yeast to DNA damage caused by basal oxidative stress. My study was focused on the regulation of Rad53 phosphorylation in different repair deficient strains to yeast. Rad53 plays decisive roles in cell cycle progression, cell death and transcriptional regulation of repair proteins to a plethora of DNA insults, including oxidative DNA damage. Rad53 activity is upregulated by phosphorylation, generating Rad53 species of various degrees of phosphorylation. I have measured steady state levels of Rad53 phosphorlyation by western blotting following SDS-polyacrylamide gel electrophoresis at different intervals in stationary phase, in various mutant backgrounds. To address the possible contribution of different repair pathways to endogenous DNA damage, I utilized two different sets of DNA repair deficient strains such as those deficient in Base excision repair (BER) and nucleotide excision repair (NER), and other set was deficient in Ku protein and NER. Interestingly, in both BERNER and Yku70rad4 strains, Rad53 phosphorylation was evident in stationary phase that is after 2 days, 4 days and 6 days but not in logarithmic phase. This covalent modification disappears after phosphatase treatment. This Rad53 modification was absent in their respective rho0 mutants, which lack mitochondrial DNA, indicating involvement of mitochondrial ROS in this checkpoint response. We analyzed mutants of different checkpoint proteins for Rad53 phosphorylation. Exclusive involvement of Rad17, Rad50 and Mec1 kinase in Rad53 phosphorylation strongly suggests processed DNA double strand breaks as critical lesions in BERNER cells. Analysis of Yku70 and NER deficient strain showed involvement of ssDNA, which is most likely at telomeres. This study consents with the model of unrepaired oxidative base damage, which can accelerate the appearance of single stranded DNA in the vicinity of double strand breaks (DSBs) or at telomeres.