Studies of Checkpoint Responses Caused by Endogenous Oxidative DNA Damage in DNA Repair Deficient Saccharomyces Cerevisiae

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dc.contributor.advisorAlvarez, Rafael
dc.contributor.committeeMemberJiang, Yiwei
dc.contributor.committeeMemberKrishnamoorthy, Raghu R.
dc.creatorPawar, Vaibhav
dc.date.accessioned2019-08-22T19:42:07Z
dc.date.available2019-08-22T19:42:07Z
dc.date.issued2008-12-01
dc.date.submitted2013-09-23T13:42:07-07:00
dc.description.abstractPawar, 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.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/20.500.12503/26757
dc.language.isoen
dc.provenance.legacyDownloads0
dc.subjectGenetic Phenomena
dc.subjectGenetics
dc.subjectGenetics and Genomics
dc.subjectGenetic Structures
dc.subjectLife Sciences
dc.subjectMedical Genetics
dc.subjectMedical Sciences
dc.subjectMedicine and Health Sciences
dc.subjectMicrobiology
dc.subjectOther Genetics and Genomics
dc.subjectCheckpoint responses
dc.subjectendogenous oxidative DNA damage
dc.subjectDNA repair
dc.subjectsaccharomyces cerevisiae
dc.subjectyeast
dc.subjectbasal oxidative stress
dc.subjectRad53
dc.subjectphosphorylation
dc.subjectwestern blotting
dc.subjectbase excision repair
dc.subjectnucleotide excision repair
dc.subjectdouble strand breaks
dc.subjecttelomeres
dc.titleStudies of Checkpoint Responses Caused by Endogenous Oxidative DNA Damage in DNA Repair Deficient Saccharomyces Cerevisiae
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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