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dc.contributor.advisorRafael Alvarez
dc.creatorChang, Woo-Jin
dc.date.accessioned2019-08-22T19:28:51Z
dc.date.available2019-08-22T19:28:51Z
dc.date.issued2001-11-01T00:00:00-08:00
dc.date.submitted2013-06-27T15:00:08-07:00
dc.identifier.urihttps://hdl.handle.net/20.500.12503/25814
dc.description.abstractChang, Woo-Jin, Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB, Doctor of Philosophy (Microbiology and Immunology), November, 2001, 92 Pages, 20 figures, 3 schemes, and bibliography. Poly(ADP-ribose) polymerase (PARP-1, E.C. 2.4.2.30) is a constitutively expressed nuclear enzyme. It comprises about 1% of the total nuclear protein and in phylogenetically well conserved in most eukaryotes, with a notable exception in yeast. PARP-1 post transitionally modifies DNA-binding proteins by transferring the ADP-ribose moiety from BNAD+. Although the exact biological function of poly(ADP-ribosyl)ation has not been clearly elucidated, the process is thought to be involved in DNA repair, replication, and gene expression. Previous studies have indicated that PARP-1 participates in eukaryotic gene expression including the genes under the control of nuclear factor-kB (NF-kB). It has been demonstrated that PARP-1 deficient mice are more resistant to lipopolysaccharide-induced endotoxic shock than isogenic wild-type mice due to the inactivation of NP-kB in the mutants. In order to further analyze the interactions between PARP-1, NF-kB, and its consensus DNA in a cell-free system, we co-incubated recombinant PARP-1 protein and the p50-subunit of NF-kB (NF-kB-p50) in the absence of DNA strand-breaks. Electrophoretic mobility shift assays (EMSA) showed that sequence-specific DNA-binding of NF-kB-p50 was dependent on autopoly(ADP-ribosyl)ation of PARP-1. The NF-kB-p50 DNA-binding was inhibitied when PARP-1 was not auto-poly(ADP-ribosyl)ated either in the absence of BNAD+ or in the presence of 3-aminobenzamide, an enzymatic inhibitor of PARP-1. Coimmunoprecipation and immunoblot analysis demonstrated that NF-kB-p50 formed a heterodimer with PARP-1 when PARP-1 was not auto-poly(ADP-ribosyl)ated. In addition, poly(ADP-ribosyl)ation assays showed that NF-kB-p50 protein was not susceptible to poly(ADP-ribosyl)ation under normal incubation conditions. Those in vitro observations described above were confirmed by experiments utilizing HeLa nuclear extracts. EMSA showed that NF-kB DNA-binding was inhibited in 3-AB-pre-treated HeLa cells. To our knowledge, this is the first report demonstrating that auto-poly(ADP-ribosyl)ation reaction by PARP-1 reversibly regulates the function of a transcription factor by inhibiting the formation of heterodimer between PARP-1 and a transcription factor.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.subjectBiomechanics and Biotransport
dc.subjectBiomedical Engineering and Bioengineering
dc.subjectCell and Developmental Biology
dc.subjectCell Biology
dc.subjectCells
dc.subjectCellular and Molecular Physiology
dc.subjectGenetics and Genomics
dc.subjectLife Sciences
dc.subjectMedical Cell Biology
dc.subjectMedical Genetics
dc.subjectMedicine and Health
dc.subjectMedicine and Health Sciences
dc.subjectDNA
dc.subjectNuclear factor kB
dc.subjectauto-poly(ADP-ribosyl)ation
dc.subjectPARP-1
dc.subjecttranscription factor
dc.titleAutomodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB
dc.typeDissertation
thesis.degree.departmentGraduate School of Biomedical Sciences
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.grantorUniversity of North Texas Health Science Center at Fort Worth
thesis.degree.nameDoctor of Philosophy
dc.contributor.committeeMemberP. Matthew
dc.contributor.committeeMemberRonald H. Goldfarb
dc.type.materialtext
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