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  • 1. Bhadra, Sankhadip Potential role of TTT complex in regulating DNA replication checkpoint in the fission yeast Schizosaccharomyces pombe

    Doctor of Philosophy (PhD), Wright State University, 2024, Biomedical Sciences PhD

    DNA replication can be perturbed by various agents that slow or stall the replication forks, causing replication stress. If undetected, stressed forks may collapse, causing mutagenic DNA damage or cell death. In response to replication stress and DNA damage, the eukaryotic cell activates the DNA replication checkpoint (DRC) and DNA damage checkpoint (DDC) pathways to promote DNA synthesis, repair, and cell survival. The two cell cycle checkpoint pathways are controlled by the protein sensor kinases Rad3 (hATR/scMec1) and Tel1 (hATM/scTel1) in fission yeast, although Tel1 plays a minimal role in checkpoint functions. Rad3 and Tel1 belong to a family of phosphatidylinositol-3-kinase-related kinases (PIKKs), whose stability is regulated by the heterotrimeric TTT (Tel2-Tti1-Tti2) complex. The current model suggests that the TTT complex works with Hsp90 and R2TP complex in the co-translational maturation of all PIKKs for their proper folding and stability. We have previously reported a tel2-C307Y mutant with a moderately reduced Rad3 protein level (~60% of wild-type cells). This mutation eliminates Rad3 mediated signaling in the DRC pathway but moderately reduces signaling in the DDC pathway. This result suggests that Tel2 of the TTT complex may specifically regulate the DRC pathway. In this study, we investigated this possibility by taking a genetic approach to analyze the functions of Tti1, the largest subunit of the TTT complex. We randomly mutated the tti1 gene and integrated the mutations at the genomic locus by pop-in and pop-out recombination strategy. As a result, 100 primary tti1 mutants were successfully screened, based on their increased sensitivities to hydroxyurea (HU) which depletes cellular dNTPs and/or the DNA damaging agent methyl methanesulfonate (MMS). Preliminary characterization of the primary Tti1 mutants, based on their relative sensitivities to HU, MMS or both agents, led us to focus on a collection of 24 mutants. Among the 24 mutants, DNA seq (open full item for complete abstract)
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    Committee: Yong-jie Xu M.D., Ph.D. (Advisor); Michael Leffak Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member); Quan Zhong Ph.D. (Committee Member); Michael Kemp Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Genetics; Microbiology; Molecular Biology; Pharmacology; Philosophy of Science; Toxicology

  • 2. Guduri, Yeseswi Archana Characterization of the hydroxyurea-induced cell elongation phenotype in fission yeast ssb1-20 mutant

    Master of Science (MS), Wright State University, 2024, Pharmacology and Toxicology

    Eukaryotic SSB, also known as replication protein A (RPA), is a single-stranded DNA binding protein that is highly conserved in eukaryotes. It is essential in multiple cellular functions such as DNA replication, repair, recombination, and cell cycle checkpoint signaling. Studying SSB's or RPA's checkpoint functions in cells is challenging due to its crucial role in cell survival. Exploring this possibility, we performed an extensive genetic screening of ssb1, which encodes the largest subunit of RPA, looking for nonlethal mutants that lack the checkpoint function. This screen has identified 25 primary mutants that are sensitive to genotoxins namely HU and MMS. Among these mutants, mutant ssb1-20 stood out for its unique response to genotoxins. The mutant cells significantly elongate in the presence of HU. In this study, we examined the cellular response of ssb1-20 to HU, MMS, and other DNA-damaging agents at different doses and various exposure times. Our results demonstrated that ssb1-20 exhibits a significantly increased cell elongation phenotype than wild-type cells when subjected to the treatment with HU, but not with the DNA-damaging agents. This observation suggests a unique response of the ssb1-20 mutant to the HU-induced stress. Further investigation is needed to understand the underlying molecular mechanisms. Overall, our study unveils the unique cell elongation phenotype of ssb1-20 in HU and suggests a novel function of RPA in the maintenance of genomic stability in eukaryotes.
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    Committee: Yong-jie Xu M.D., Ph.D (Advisor); Ravi P. Sahu Ph.D (Committee Member); Michael Kemp Ph.D (Committee Member) Subjects: Pharmacology; Toxicology

  • 3. Zavada, Nathen Genomic instability at a polypurine/polypyrimidine repeat sequence

    Master of Science (MS), Wright State University, 2022, Biochemistry and Molecular Biology

    Microsatellite repeat sequences have been shown to induce replication stalling, fork collapse, double-strand breaks (DSBs), and possibly stimulate break-induced replication. In this study we use a dual-fluorescent HeLa model that is designed to monitor recombination at an ectopic site through use of flow cytometry and inverse PCR with a microsatellite in the lagging strand for DNA synthesis. To test the stability of the 78 bp polypurine/pyrimidine repeat from the PDK1 locus, we subjected cells to replication stress drugs designed to induce DSBs and measure break-induced replication (BIR). The study revealed that polypurine repeat cells undergo endogenous stress contributing to instability at the ectopic site as well as slow cell growth. Further, we show that there is an orientation dependency for instability with the (Pu)78 cells being more unstable. Lastly, we present a novel candidate for a protein involved in break-induced replication, COPS2. Preliminary experiments show this protein produces unique recombination patterns when knocked down.
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    Committee: Michael Leffak Ph.D. (Advisor); Mike Kemp Ph.D. (Committee Member); Michael Markey Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Genetics; Molecular Biology

  • 4. Slayton, Mark Protein-DNA Interactions of pUL34, an Essential Human Cytomegalovirus DNA-Binding Protein

    Doctor of Philosophy (PhD), Ohio University, 2018, Molecular and Cellular Biology (Arts and Sciences)

    Human cytomegalovirus (HCMV) is primarily an opportunistic pathogen in human, causing significant disease in immunocompromised individuals. A large, double-stranded DNA genome (~230 kilobases) provides the coding capacity for over 200 genes, of which only 25% are required for viral replication in cell culture. The viral UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are essential for replication, and viruses lacking the proper expression of pUL34 cannot replicate in cell culture. Interactions of pUL34 with DNA binding sites (US3 and US9?) represses transcription of (these) two viral immune evasion genes that are dispensable for replication in cell culture. There are 12 additional predicted pUL34-binding sites present in the HCMV genome (strain AD169), with three of them concentrated near the HCMV origin of lytic replication (oriLyt). Analysis of 47 clinical isolates of HCMV confirmed that the predicted UL34-binding sites were highly conserved. Protein-DNA interactions were analyzed during infection with ChIP-seq and confirmed that pUL34 binds to the human and viral genome during infection, including at the three predicted UL34-binding sites in the oriLyt region. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated, oriLyt-dependent DNA replication. Subsequently, mutagenesis of these same sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with 3 virus proteins that are essential for viral DNA replication - IE2, UL44, and UL84, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Lastly, mutagenesis of the predicted UL34-binding site in the third exon of another essential viral gene, UL37, demonstrated that some UL34-binding sites are not important for viral replication.
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    Committee: Bonita Biegalke (Advisor); Calvin James (Committee Member); Mark Berryman (Committee Member); Justin Holub (Committee Chair) Subjects: Genetics; Molecular Biology; Virology

  • 5. Poudel, Sumeet Interaction of DUE-B and Treslin during the initiation of DNA replication

    Doctor of Philosophy (PhD), Wright State University, 2016, Biomedical Sciences PhD

    The initiation of DNA replication is a highly regulated and coordinated process. To ensure that the entire genome is replicated only once per cell cycle, many replication proteins are assembled on the chromatin in a step-wise and cell cycle dependent manner. This process is controlled by interaction of replication proteins, post-translational modifications of the replication factors, control of cellular localization of the proteins, or replication factor degradation after their function terminates. Two kinases, CDK (cyclin dependent kinase) and DDK (Dbf4/Drf1 dependent kinase), play important roles during the initiation stage of DNA replication. The c-myc DNA unwinding element-binding protein (DUE-B) is an essential replication protein that interacts with both the replicative helicase (minichromosome maintenance (MCM)) and the helicase activator (Cdc45) at mammalian replication origins, and is required to load Cdc45 onto chromatin in Xenopus egg extracts. Here, using co-immunoprecipitation experiments, I show that DUE-B interacts with the protein Treslin/TICRR (TopBP1- interacting, replication stimulating protein/TopBP1-interacting, checkpoint and replication regulator, simply refered to as Treslin) which has dual roles in replication and checkpoint in vivo. Treslin, an orthologue of yeast Sld3, is an essential CDK substrate during replication initiation and DNA damage signaling. Treslin collaborates with TopBP1 in the Cdk2-mediated loading of Cdc45 onto replication origins. The interaction between DUE-B and Treslin does not require the presence of DNA but requires TopBP1, another protein with dual functions in replication and checkpoint. TopBP1, a human homolog of budding yeast Dpb11, is a multi BRCT domain containing protein. It is a scaffolding protein that allows interaction between replication and checkpoint proteins. I show that the interaction between DUE-B and Treslin is cell cycle and checkpoint regulated. Interaction between DUE-B and Treslin increases du (open full item for complete abstract)
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    Committee: Michael Leffak Ph.D. (Advisor); Ashot Kozak Ph.D. (Committee Member); Yong-jie Xu M.D./Ph.D. (Committee Member); Steven Berberich Ph.D. (Committee Member); Scott Baird Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research

  • 6. Yao, Jianhong DUE-B, A NEW HUMAN DNA REPLICATION PROTEIN, IS THE FUNCTIONAL HOMOLOG OF S. CEREVISIAE SLD3

    Master of Science (MS), Wright State University, 2009, Biochemistry and Molecular Biology

    DNA unwinding elements (DUEs) are commonly found at DNA replication origins. The DUE binding protein (DUE-B) is crucial for the initiation of DNA replication in eukaryotes. The unique 59 amino acid C-terminal part of DUE-B shares nearly 50% similarity with yeast the C-terminus of Sld3. DUE-B plays a key role in eukaryotic DNA replication because it is required for the loading of Cdc45, the MCM helicase activator, on chromatin. Here we show that DUE-B, just like yeast Sld3, binds to Cdc45 and TopBP1 through its C-terminus in Sf9 cells and in vitro. We also show that DUE-B, Cdc45 and TopBP1 form a heterotrimeric complex in vitro. The mass spectrometric data show that dominant negative Sf9 DUE-B is not phosphorylated but functional HeLa DUE-B is phosphorylated. All these data suggest that human DUE-B is a functional homolog of yeast Sld3.
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    Committee: Michael Leffak (Advisor); Steven Berberich (Committee Member); John Paietta (Committee Member) Subjects: Biomedical Research

  • 7. Kemp, Michael Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B

    Doctor of Philosophy (PhD), Wright State University, 2006, Biomedical Sciences PhD

    Duplication of the genome during S phase of the mitotic cell cycle begins at thousands of sites along chromosomes termed origins of replication. Although many of the essential protein components catalyzing events at these sites are known and are conserved throughout eukaryotes, the likelihood or efficiency of initiation of DNA synthesis at any given genomic site is expected to be influenced by other novel factors, including aspects of chromatin and DNA structure. Here I show that increased histone H4 acetylation at replication origin loci occurs after treatment with the histone deacetylase inhibitor TSA and coincides with a loss of specific initiation site selection both within origin loci and throughout the genome. Furthermore, new replication initiation sites become activated or used with greater frequency after treatment with TSA, and TSA promotes the activation of replication origins earlier during the S phase of the cell cycle. These data suggest a physiological role for histone acetylation in controlling the initiation of DNA synthesis at specific chromosomal sites. Regions of helically unstable DNA termed DNA unwinding elements (DUEs) are commonly found at replication origins, and our laboratory identified a DUE-binding protein (DUE-B) using the c-myc DUE in a yeast one-hybrid screen. Here I demonstrate that DUE-B is required for efficient entry into S phase in human cells and for efficient replication in the Xenopus egg extract replication system. Structural analyses show the N-terminal portion of the protein to be identical to that of bacterial D-aminoacyl-tRNA deacylases. Human DUE-B possesses this function in vitro and the ability to hydrolyze ATP, suggesting that DUE-B may be a multifunctional esterase. Unique to vertebrate homologs of DUE-B is a C-terminal extension of 62 amino acids that binds DNA and is targeted for phosphorylation by CK2. The addition of the C-terminal domain to DUE-B in higher eukaryotes may have coincided during evolution with the (open full item for complete abstract)
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    Committee: Ira Leffak (Advisor) Subjects:

  • 8. Shanahan, Matilyn Instability and Extrachromosomal Circular DNA Formation at Microsatellites and Unstable DNA Sequences

    Master of Science (MS), Wright State University, 2022, Biochemistry and Molecular Biology

    We have previously documented our evidence of genetic instabilities at the (Pu/Py)78 and (ATTCT)47 sequences and our reasoning for identifying break-induced replication (BIR) as the mode of repair responsible for the mutations in the DNA flanking the unstable inserts. Now, as our lab investigates the protein mechanisms at play in the BIR pathway taking place at these sites, we are also expanding our knowledge of how this mechanism extends into the pathways responsible for forming extrachromosomal circular DNA (eccDNA) molecules. We have documented the phenomena posed as the driving factors for eccDNA formation in our systems containing (Pu/Py)78 and (ATTCT)47. Therefore, in this defense, we worked to uncover how STN1, COPS2, Pol and, generally, BIR function to produce eccDNAs in mammalian cells containing these unstable DNA sequences using inverse PCR (iPCR) to detect circular DNAs being generated in the genomic contents of these cells before and after knockdown of the proteins of interest.
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    Committee: Michael Leffak Ph.D. (Advisor); Kwang-Jin Cho Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

  • 9. Abrefa, Darlington Genetic Study of Checkpoint Defects of the Mus81-1 Mutant in the Fission Yeast Schizosaccharomyces Pombe.

    Master of Science (MS), Wright State University, 2019, Microbiology and Immunology

    In response to various perturbations of DNA replication, the DNA replication checkpoint is activated in eukaryotes to stimulate a cascade of cellular responses that are crucial for maintaining genome stability and cell survival. Defects in the checkpoint pathway result in mutations and genome instability, which is a hallmark for cancers. This study used a genetic approach to identify a mutation in the MMS (methyl methanesulfonate) and UV-sensitive protein Mus81, a DNA repair enzyme that resolves aberrant DNA structures through the homologous recombination pathway. We show that a single missense mutation, identified in fission yeast mus81-1, causes moderate reduction in the phosphorylation levels of the major DNA replication checkpoint proteins Mrc1(Claspin) and Cds1(Chk2) in fission yeast. We also show that the mutation directly affects the DNA repair and the DNA damage checkpoint mediated by Chk1 that causes dramatic cell lethality in mus81-1 mutant upon treatment with the DNA damaging agents: MMS, UV and Bleomycin.
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    Committee: Yong-jie Xu M.D., Ph.D. (Advisor); Michael G. Kemp Ph.D. (Committee Member); Dawn P. Wooley Ph.D. (Committee Member); Nancy J. Bigley Ph.D. (Committee Member) Subjects: Genetics; Microbiology; Molecular Biology

  • 10. Perl, Abbey Leveraging Small Molecule Activators of Protein Phosphatase 2A (PP2A) to Elucidate PP2As Role in Regulating DNA Replication and Apoptosis

    Doctor of Philosophy, Case Western Reserve University, 2020, Pharmacology

    Aberrant signal transduction resulting from dysregulated phosphorylation is a hallmark of human cancer. Altered phosphorylation has broad implications on cancer biology. Much work has been done characterizing the effects of individual kinases with their cancer phenotypes. However, the structural complexity of their counterparts, phosphatases, has limited our knowledge of these signaling events. Protein Phosphatase 2A (PP2A), one such negative regulator of multiple oncogenic kinases, has been well characterized as a tumor suppressor protein that when inhibited can lead to cellular transformation. PP2A is a heterotrimeric complex whose substrate specificity is dependent on one of 23 different regulatory subunits that can bind to form over 60 distinct holoenzyme complexes. Although PP2A's function as a general tumor suppressor is well studied, the role of PP2A on specific tumor suppressive signaling pathways and the specific holoenzymes mediating this signaling are not completely understood. Through chemical and genetic approaches, this work characterizes a new role for PP2A in the regulation of DNA replication, and links PP2A effects on replication with its ability to induce apoptosis. 2 Utilizing both a gain of function chemical biology approach and loss of function genetic approaches to modulate PP2A activity, we demonstrate that increasing PP2A activity can interrupt ongoing DNA replication resulting in a collapse of replication forks, the induction of double-stranded DNA (dsDNA) breaks, and a replication stress response that is PP2A dependent. Additionally, we show that increasing PP2A activity during replication causes a dissociation of the replisome, a common mechanism of inhibiting ongoing replication. Furthermore, patients harboring mutations in PP2A are shown to have a higher fraction of their genome altered, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. Moreover, knockdown of the (open full item for complete abstract)
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    Committee: Goutham Narla M.D./Ph.D. (Advisor); Derek Taylor Ph.D. (Committee Chair); John Mieyal Ph.D. (Committee Member); Youwei Zhang Ph.D. (Committee Member); Amar Desai Ph.D. (Committee Member) Subjects: Biomedical Research; Cellular Biology; Pharmacology

  • 11. Tokarsky, E. John Analysis of Human Y-Family DNA Polymerases and PrimPol by Pre-Steady-State Kinetic Methods

    Doctor of Philosophy, The Ohio State University, 2018, Biophysics

    Eukaryotic genomic DNA is efficiently and accurately replicated to ensure that an exact copy is created before cell division occurs. The complex machinery involved in DNA replication is tightly coordinated and regulated to ensure it proceeds in a relatively uninhibited manner. The enzymes responsible for copying the genome are known as DNA polymerases and these are responsible for catalyzing nucleotidyl transfer of the building blocks of DNA, deoxyribonucleotides (dNTPs), onto growing primer strands in the 5'-3' direction. The active sites of DNA polymerases allow them to facilitate template-dependent nucleotidyl transfer based on Watson-Crick base pairing rules, i.e. adenine:thymine and cytosine:guanine (A:T and C:G). In humans, these enzymes must proceed at an extremely fast rate in order to replicate approximately 6 billion base pairs during each cell cycle. Reactive hydrocarbons, high energy UV-light, or free radicals generated during cellular processes (i.e. electron transport chain), modify DNA bases that can cause DNA polymerases to stall. Specialized DNA polymerases, from the Y-family, catalyze translesion DNA synthesis to replicate through modified DNA bases in order for the replication machinery to continue efficient DNA synthesis. Y-family DNA polymerases are able to accommodate bulky, modified bases into their active sites because they are flexible, and solvent-exposed. This characteristic makes them perfect candidates to bypass many types of DNA damage. However, these flexible active sites make them error-prone and thus, Y-family DNA polymerases must be tightly regulated to ensure that high levels of DNA mutations that lead to genetic disease, are not introduced. In this dissertation, I will describe my work with four human Y-family DNA polymerases, Eta, Kappa, Iota, Rev1, and their abilities to bypass an air pollution-generated, bulky DNA lesion. 3-nitrobenzanthrone (3-NBA) is a byproduct of diesel fuel combustion that binds to particulate m (open full item for complete abstract)
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    Committee: Zucai Suo Ph.D. (Advisor); Jeff Kuret Ph.D. (Committee Chair); Charles Bell Ph.D. (Committee Member); Zhengrong Wu Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biophysics

  • 12. Stephenson, Anthony Mechanistic studies of enzymes involved in DNA transactions

    Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

    DNA transactions (i.e. enzymatic reactions that copy and modify DNA) are central in the functions of life. Several housekeeping enzymes are responsible for the maintenance of DNA in cells. Among these enzymes, DNA polymerases are crucial. However, various types of DNA polymerases exist and it is imperative that the optimal types are recruited for each particular DNA transaction. For example, replicative DNA polymerases have evolved to copy DNA with high fidelity. Replicative DNA polymerases are very sensitive to the structure of DNA and do not function properly with damaged DNA substrates. Hence, other types of DNA polymerases, optimized for particular types of DNA damage, have evolved to utilize specific types of commonly occurring damaged DNA substrates. Among these specialized enzymes are the X-family DNA repair polymerases which includes DNA polymerase lambda. The primary function of DNA polymerase lambda is to perform end processing during DNA strand break repair. DNA polymerase lambda is specialized for filling in gaps between the broken DNA ends. However, with specialization for damaged DNA substrates often comes a sacrifice of fidelity due to structural changes of the active site to accommodate damaged DNA structures. Thus, DNA repair polymerases must be controlled to prevent inappropriate recruitment during DNA replication, which could result in deleterious mutations. DNA repair polymerases contain accessory domains, in addition to the typical polymerase domains, which may provide means of regulating recruitment of these enzymes to particular DNA repair complexes. Chapter 2 in this dissertation explores the potential regulatory functions of the accessory domains of DNA polymerase lambda for recruitment to DNA repair complexes using comparative cellular biology techniques including fluorescence-based imaging, western blotting, sub-cellular fractionation, and cytotoxicity assays. When DNA repair mechanisms fail, DNA can be compromised via mutation resulting (open full item for complete abstract)
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    Committee: Zucai Suo Ph.D. (Advisor); Jian-Qiu Wu Ph.D. (Committee Member); Dmitri Kudryashov Ph.D. (Committee Member); Harold Fisk Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Cellular Biology

  • 13. Raper, Austin Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing

    Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

    DNA acts as a molecular blueprint for life. Adenosine, cytidine, guanosine, and thymidine nucleotides serve as the building blocks of DNA and can be arranged in near-endless combinations. These unique sequences of DNA may encode genes that when expressed produce RNA, proteins, and enzymes responsible for executing diverse tasks necessary for biological existence. Accordingly, careful maintenance of the molecular integrity of DNA is paramount for the growth, development, and functioning of organisms. However, DNA is damaged upon reaction with pervasive chemicals generated by normal cellular metabolism or encountered through the environment. The resulting DNA lesions act as roadblocks to high-fidelity A- and B-family DNA polymerases responsible for replicating DNA in preparation for cell division which may lead to programmed cell death. Additionally, these lesions may fool the polymerase into making errors during DNA replication, leading to genetic mutations and cancer. Fortunately, the cell has evolved DNA damage tolerance as an emergency response to such lesions. During DNA damage tolerance, a damage-stalled high-fidelity polymerase is substituted for a specialized Y-family polymerase, capable of bypassing the offending DNA lesion, for replication to continue. However, the ability of the specialized polymerase to bypass DNA lesions occurs at the expense of replication fidelity. Hence, tight regulation of polymerase exchange during DNA damage tolerance is imperative to ensure timely bypass of a lesion by the Y-family member, as well as prompt polymerase replacement by an A- or B-family member to limit DNA replication errors (i.e. mutations). Nevertheless, mistakes during DNA damage tolerance that evade DNA repair pathways are intimately connected to mutagenesis and may lead to cancer or numerous other genetic diseases. Until recently, making corrections to erroneous DNA sequences in the cell was prohibitively time-consuming, expensive, and laborious. However, the (open full item for complete abstract)
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    Committee: Zucai Suo Ph.D. (Advisor); Zucai Suo Ph.D. (Advisor); Richard Swenson Ph.D. (Committee Chair); Richard Swenson Ph.D. (Committee Chair); Ross Dalbey Ph.D. (Committee Member); Ross Dalbey Ph.D. (Committee Member); Michael Poirier Ph.D. (Committee Member); Michael Poirier Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Chemistry; Molecular Biology; Molecular Chemistry

  • 14. Wallenmeyer, Petra Investigating Current Mechanistic Models of DNA Replication and Repair

    Master of Science, The Ohio State University, 2017, Chemistry

    DNA polymerases (pols) play a pivotal role in both the replication and the repair of genomic DNA. Replicative pols are highly accurate and processive, synthesizing long stretches of DNA in a single binding event, while repair and bypass pols are error-prone and only able to incorporate a few nucleotides before dissociation. During replication, the pol may encounter DNA modifications induced by endogenous and exogenous factors such as oxidative metabolites, UV radiation, or epigenetic additions. These modifications may alter the local structure of DNA, resulting in inhibition of the replicative pol and stalling of the replication machinery. When the replicative pol stalls, a repair or bypass pol can take over and perform translesion synthesis (TLS). During TLS, a nucleotide is inserted opposite a lesion on the template DNA strand before the replicative pol can continue DNA synthesis. The mechanistic details of DNA replication, bypass, and repair are areas of ongoing research and are important to other areas of research such as drug design, cancer research, metabolism, and aging. The overarching goal of my research was to contribute to the mechanistic understanding of how pols perform DNA synthesis and bypass of DNA lesions. With this goal in mind, one of my main projects was to investigate the bypass kinetics of a common epigenetic signal, modification of the C5-position on cytosine (5xC). I used a specialized pol, human pol iota, to conduct this investigation. Using pre-steady state kinetic methods, I determined the dissociation constant (Kd) and maximum incorporation rate (kpol) for each deoxynucleoside (dNTP) opposite each 5xC modification. I also attempted to determine the structural details of the two accessory subunits of human pol epsilon, a replicative DNA pol that carries out leading strand synthesis, via X-ray crystallography to propose a hypothesis for its intrinsically high fidelity and processivity. Other projects I was involved in included investiga (open full item for complete abstract)
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    Committee: Zucai Suo Dr. (Advisor); James Cowan Dr. (Committee Member); Thomas Magliery Dr. (Committee Member) Subjects: Biochemistry; Chemistry

  • 15. Gadkari, Varun A Multi-Disciplinary Investigation of Essential DNA Replication Proteins

    Doctor of Philosophy, The Ohio State University, 2017, Biochemistry Program, Ohio State

    An organism's DNA is constantly under attack from various exogenous and endogenous DNA damaging agents. Thus, to assure survival, all living cells have evolved to maintain the genetic integrity of their DNA by various pathways. If left unrepaired, DNA damage sites, or “lesions” can block DNA replication by stalling DNA polymerases, the enzymes responsible for DNA replication. Ultimately, if a stalled replication fork is not rescued, the cell will undergo apoptosis. To bypass DNA lesions, organisms in all domains of life initiate a process known as Translesion DNA Synthesis (TLS). During TLS, the stalled replicative DNA polymerase is displaced by specialized Y-family DNA polymerases that are capable of efficiently bypassing various forms of DNA damage. While Y-family DNA polymerases are proficient in TLS, the fidelity of the process is a notable cause for concern. TLS mechanisms of the different Y-family DNA polymerases vary greatly, and often introduce mutations in the DNA which can lead to carcinogenesis. Thus, the activity of Y-family DNA polymerases must be strictly regulated. To this end all living organisms depend on evolutionarily conserved sliding DNA clamps which bind the DNA in a toroidal fashion, and slide along DNA during replication, serving as a scaffold for the DNA replication and repair machinery. During replication fork progression, the DNA clamp can regulate the various enzymatic activities by binding multiple proteins simultaneously as they process DNA. The overarching goal of my research has been to establish how the mechanisms of DNA replication, and TLS affect DNA clamp mediated polymerase switching (pol switching) to allow for efficient DNA replication while also preventing DNA mutagenesis. My working hypothesis is that the DNA clamp is bound to both replicative and TLS DNA polymerases at the replication fork, and that pol switching is governed by differences in DNA replication efficiency. Upon encountering a DNA lesion, replicative DNA pol (open full item for complete abstract)
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    Committee: Zucai Suo Ph.D. (Advisor); Jane Jackman Ph.D. (Committee Member); Comert Kural Ph.D. (Committee Member); Richard Swenson Ph.D. (Committee Member) Subjects: Biochemistry

  • 16. Gadgil, Rujuta Yashodhan Instability at Trinucleotide Repeat DNAs

    Master of Science (MS), Wright State University, 2016, Biochemistry and Molecular Biology

    Trinucleotide repeats (TNRs) are sequences prone to formation of non-B DNA structures and mutations; undergo expansions in vivo to cause various inherited neurodegenerative diseases. Hairpin structures formed during DNA replication or repair can cause replication fork stalling and if left unrepaired could cause single or double strand DNA breaks. To test and study this hypothesis we have devised a novel two color marker gene assay to detect DNA breaks at TNRs. By inducing replication stress our results show that TNRs are prone to DNA strand breaks and it is dependent on the repeat tract length. Double strand breaks at structured DNA are repaired differently than `clean' DSBs. The cells which undergo breaks die off, possibly due to inability to repair breaks. Translesion polymerases help tolerate DNA damage at TNR region.
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    Committee: Michael Leffak Ph.D. (Advisor); John Paietta Ph.D. (Committee Member); Michael Markey Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

  • 17. Maxwell, Brian Multi-disciplinary Investigation of the Kinetics and Protein Conformational Dynamics of DNA Replication and Oxidative DNA Damage Bypass and Repair

    Doctor of Philosophy, The Ohio State University, 2014, Biophysics

    Genomic DNA is under constant attack from both environmental sources as well as toxic byproducts of natural cellular processes. Cells therefore must utilize a variety of pathways to repair or otherwise cope with damaged DNA in order to survive and to faithfully copy the genome during cell division. Unrepaired DNA damage sites or “lesions” can act as roadblocks to DNA replication by stalling the cell's replicative DNA polymerase, a molecular motor responsible for copying the genome. In order to avoid cell death due to stalled DNA replication, DNA lesions can be bypassed through DNA translesion synthesis (TLS) whereby a stalled high-fidelity replicative DNA polymerase is replaced by a specialized lesion-bypass DNA polymerase capable of bypassing the damaged site. However, these specialized DNA lesion-bypass polymerases are much more error prone during DNA replication than replicative DNA polymerases, and mistakes made in copying the DNA during this process can potentially lead to cancer formation. Therefore polymerase switching during TLS must be tightly regulated to minimize the introduction of mutations while still allowing for TLS. In addition, cells also utilize various enzymes in the Base Excision Repair (BER) pathway to remove DNA lesions prior to the replication of DNA in order to avoid the potential mistakes that could be made during TLS. The goal of my research has been to establish an in depth understanding of the molecular mechanisms of the replication of damaged and undamaged DNA and of the BER pathway using a variety of in vitro biochemical and biophysical tools. My working hypothesize is that, in order to regulate polymerase switching and DNA damage repair, DNA damage sites can alter various properties of DNA polymerases and the enzymes involved in BER including how they bind to DNA, the rate at which they perform their catalytic functions and how they change their structures during the course of the reaction. In order to test this hypothesis I have u (open full item for complete abstract)
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    Committee: Zucai Suo PhD (Advisor); Ralf Bundschuh PhD (Committee Member); Richard Swenson PhD (Committee Member) Subjects: Biophysics

  • 18. ROBISON, JACOB INTERACTION OF THE Mre11/Rad50/Nbs1 (MRN) COMPLEX AND REPLICATION PROTEIN A (RPA) IN RESPONSE TO DNA DAMAGE

    PhD, University of Cincinnati, 2005, Medicine : Toxicology (Environmental Health)

    Both replicative stress and DNA damage initiate cellular processes collectively termed the DNA damage response. These processes include activation of appropriate DNA repair mechanisms, cell cycle checkpoints, and in some cases, apoptosis. Accurate and efficient operation of the DNA damage response is essential for preventing mutations that may lead to oncogenic transformation or some types of inherited diseases. The DNA damage response involves sensing the damage, activation of specific kinases that transduce the activation signal via protein phosphorylation, and activation of effector proteins that carry out the functional aspects of the response. Two hallmarks of the DNA damage response are phosphorylation of key regulatory proteins and aggregation of multiprotein complexes into foci at or near the site of damage. The proteins that are phosphorylated and the composition of the foci depend upon the nature of the DNA lesion, and changes as the damage is recognized, processed and then repaired. Although different types of DNA damage activate specific repair proteins and pathways, some proteins respond to multiple types of lesions. Two protein complexes essential for the response to many lesions types are the Mre11/Rad50/Nbs1 (MRN) complex and replication protein A (RPA). Evidence supports the hypothesis that both of these complexes have multiple roles in the DNA damage response, including initial DNA damage recognition, activation of the signal transducing kinases and functional roles in DNA repair pathways. Although the MRN complex and RPA both become phosphorylated and form foci in response to multiple types of DNA lesions, we found that they co-localize to nuclear foci only in response to a subset of lesions. However, depletion of RPA via siRNA abrogates the ability of the MRN complex to form foci. These data suggest that the MRN complex and RPA have functional activities that can be both dependent and independent of each other. Understanding the determinant of wh (open full item for complete abstract)
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    Committee: Dr. Kathleen Dixon (Advisor) Subjects:

  • 19. Boutemy, Laurence Analysis of the Interactions between the 5' to 3' Exonuclease and the Single-Stranded DNA-Binding Protein from Bacteriophage T4 and Related Phages

    Doctor of Philosophy, University of Toledo, 2008, Chemistry

    DNA replication and repair is one of the most important cellular processes, since preserving the integrity of the DNA genome is essential to all forms of life. Many proteins are involved in the DNA replication process, and their interaction ensures that the DNA is duplicated and repaired in a coordinated and efficient manner. Bacteriophage T4 is a very good model to study DNA replication, since it encodes all the proteins required at the replication fork, proteins which have been extensively characterized. However, how these proteins interact and coordinate the replication process is still largely unknown. One of these interactions that appears to govern the rate and efficiency of the lagging strand synthesis occurs between the 5' to 3' exonuclease RNase H and the single-stranded DNA-binding 32 protein. The interaction between these two proteins is the focus of this work. RNase H and the 32 protein, as well as a number of mutants and truncations, were cloned, expressed and purified. These proteins were then used to form different variants of the RNase H + 32 protein complex, which were characterized through biophysical and structural studies. A crystal structure was obtained for the RNase H + 32-B truncation. This structure, along with the results obtained from the biophysical experiments, provides valuable information on how these two proteins interact to coordinate the lagging strand DNA replication. c Finally, the study of the interaction between RNase H and the 32 protein from bacteriophage Rb 69, a phage related to bacteriophage T4, was also initiated.
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    Committee: Timothy Mueser PhD (Advisor); Max Funk PhD (Committee Member); Ronald Viola PhD (Committee Member); Hermann Von Grafenstein PhD (Committee Member) Subjects: Biochemistry

  • 20. Senger, Anne A Study of DNA Replication and Repair Proteins from Bacteriophage T4 and a Related Phage

    Master of Science, University of Toledo, 2004, Chemistry

    In the Mueser laboratory, we study how DNA replication and repair proteins recognize DNA in a structure-specific manner. Bacteriophage T4 is used as a model system to study DNA replication as it encodes all ten proteins required for DNA replication. Much is known about how the individual proteins function in replication but not much is known about the structural aspects of the protein-protein or protein-DNA interactions at the replication fork. The goal of our research is to study how these replication proteins interact with each other and with DNA. We work towards achieving this goal by crystallizing the protein-protein and protein-DNA complexes and then solving their structures, using macromolecular crystallography techniques. We then use the structural information gathered to analyze the interactions. The overall goal of this master's thesis project was to learn many of the techniques involved in protein chemistry and protein crystallization. My research was tailored to protein expression, purification and crystallization so I could learn an array of techniques and become familiar with various pieces of instrumentation. I wanted to be able to use this knowledge in future research positions. My work was focused on two of the replication proteins from Bacteriophage T4: T4 gene 59 helicase assembly protein and T4 gene 32 single-stranded binding protein. These two proteins interact in the absence of DNA and form a complex at the replication fork. I was responsible for expressing mutated and truncated forms of the native proteins on a large scale and developing purification protocols in order to prepare pure protein for crystal screening. After my research with the T4 helicase assembly protein began, I also started working on a similar helicase assembly protein from a related system – bacteriophage KVP40 59 protein. I was also responsible for developing a purification protocol for single-stranded DNA substrates that were used to prepare forked substrates for the cryst (open full item for complete abstract)
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    Committee: Timothy Mueser (Advisor) Subjects: Chemistry, Biochemistry