Browsing by Subject "yeast"
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Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.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.