Protein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B

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dc.contributor.advisorAlvarez, Rafael
dc.contributor.committeeMemberBen S. Aar…
dc.contributor.committeeMemberWu, Mingehi
dc.creatorConfer, Nils Forgard
dc.date.accessioned2019-08-22T21:31:25Z
dc.date.available2019-08-22T21:31:25Z
dc.date.issued2003-12-01
dc.date.submitted2013-06-27T15:00:08-07:00
dc.description.abstractConfer, 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.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/20.500.12503/29395
dc.language.isoen
dc.provenance.legacyDownloads0
dc.subjectBiomedical Engineering and Bioengineering
dc.subjectCell and Developmental Biology
dc.subjectCell Biology
dc.subjectCells
dc.subjectEnzymes and Coenzymes
dc.subjectGenetic Phenomena
dc.subjectGenetic Processes
dc.subjectGenetic Structures
dc.subjectInvestigative Techniques
dc.subjectLife Sciences
dc.subjectMedical Cell Biology
dc.subjectMedical Genetics
dc.subjectMedicine and Health Sciences
dc.subjectSurvival Analysis
dc.subjectCell survival
dc.subjectcell death
dc.subjectpoly(ADP-ribose) polymerase-1
dc.subjectDNA Polymerase B
dc.subjectProtein-Protein Interactions
dc.subjectbase excision repair
dc.titleProtein-Protein Interactions Between Poly(ADP-Ribose) Polymerase-1 and DNA Polymerase B
dc.typeDissertation
dc.type.materialtext
thesis.degree.departmentGraduate School of Biomedical Sciences
thesis.degree.disciplineBiomedical Sciences
thesis.degree.grantorUniversity of North Texas Health Science Center at Fort Worth
thesis.degree.nameDoctor of Philosophy

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