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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. Tcyrulnikov, Nikolai Pyridinium Salts: from Photoinduced Through-Space Electron Delocalization to Novel Spontaneous Reactions Causing Thermal DNA Damage

    Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Photochemical Sciences

    The first chapter of this dissertation is devoted to the investigation of the pimerization and photo-induced electron-transfer processes in the series of bis(pyridinium) alkane salts and covalently linked monoquat. The DFT analysis of bis(pyridinium) alkane systems allowed us to compare the energies of the different conformations of dications and radical cations of these salts formed upon single electron reduction which is important for the determination of the conformations that favor the formation of the dimer radical cation. The results of these calculations made an important contribution to the studies of pimerization in bis(pyridinium)alkane salts. Reduction of monomeric and covalently linked monoquat (1-methyl-4-(4-pyridyl)pyridinium) was assessed experimentally by means of Cyclic Voltammetry and transient absorption spectroscopy. A number of interesting observations were made to provide a basis for the improved design of novel photochemical DNA damaging agents that contain electron-poor pyridinium or monoquat `arms' able to undergo pimerization process. The second part of the dissertation covers the spontaneous aerobic catalyst-free transition from 1,1,2,2-tetrakis(N-methylpyridin-4-ium)ethane iodide to the corresponding epoxide (major product) and alkene (minor product). This reaction represents a rare transition from a substituted alkane to the epoxide. The mechanism of the reaction was proposed based on the intermediates and products characterization and further supported by the kinetic modeling. It was demonstrated that the oxidation proceeds through the formation of the air sensitive monomethine cyanine dye dimer. This reaction intermediate was involved in the production of the Reactive Oxygen Species (ROS) that are known to induce DNA damage. In light of this observation, the DNA toxicity of 1,1,2,2-tetrakis(N-methylpyridin-4-ium)ethane under aerobic aqueous conditions was examined. The results of this study are presented in the last chapter of th (open full item for complete abstract)
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    Committee: R. Marshall Wilson Dr. (Advisor); Pavel Anzenbacher Dr. (Committee Member); Hong Lu Dr. (Committee Member); Kit Chan Dr. (Other) Subjects: Biochemistry; Chemistry; Organic Chemistry; Physical Chemistry

  • 3. Chakraborty, Debanjana Molecular Basis of Ultrafast Catalysis by DNA Photolyase

    Doctor of Philosophy, The Ohio State University, 2023, Biochemistry Program, Ohio State

    Cyclobutane pyrimidine dimers (CPD) are carcinogenic DNA photolesions formed between two adjacent thymine bases in genomic DNA by UV irradiation. DNA photolyases are ancient photoenzymes that evolved to specifically recognize, bind to and repair these lesions using blue light in ultrafast timescale. Comprehensive molecular level understanding of enzymatic CPD cycloreversion is lacking. We highlight various molecular factors from both enzyme active site and substrate itself, that modulate various electron transfer (ET) and bond rearrangement steps involved in the repair photocycle and determine the overall quantum repair yield. We tracked the real time repair dynamics of carefully planned enzyme and substrate variants using femtosecond spectroscopy. We found that active site amino acids modulate solvation around the cofactor-lesion pair and influence associated ET parameters. Steric and electrostatic properties of the substrate and the enzyme active site dictate the dimer splitting yield by tuning bond cleavage and futile ET steps. Finally, we found that the photolyases have evolved to fit the lesion in a specific binding configuration to maximize electronic coupling with electron donating cofactor and selectively destabilize various intermediates to achieve modest to high repair yields.
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    Committee: Dongping Zhong (Advisor) Subjects: Biochemistry; Biophysics; Chemistry

  • 4. Alhawach, Venicia DNA Instability at (ATTCT) Microsatellites in Human Cells

    Doctor of Philosophy (PhD), Wright State University, 2023, Biomedical Sciences PhD

    Microsatellites are short tandem repeats of DNA (1-9 nucleotides) that are unstable in the genome. They can expand and contract which leads to mutations and rearrangements that jeopardize genomic integrity. When encountered by a replication fork, repeats of these microsatellites can form non-B DNA structures which cause replication forks to stall and collapse eventually leading to double stranded breaks (DSBs) and replication stress. One form of repair of replication dependent DSBs happens via break induced replication (BIR), a highly mutagenic repair pathway. The main objective of this study is to understand how ATTCT pentanucleotide repeats affect replication and chromosomal instability through quantitively measuring replication dependent DSBs at these microsatellites and by investigating recombination signatures generated at these microsatellites after repair. Expansion of ATTCT pentanucleotide repeats at the ataxin 10 locus is responsible for the human disease spinocerebellar ataxia type 10 (SCA10). Therefore, we focus on three aims. Aim 1: To determine the effect of expanded ATTCT repeats on genome stability when functionally replacing the DNA unwinding element (DUE) of the c-myc origin of replication. In a novel dual fluorescence reporter gene system, we use flow cytometry, inverse PCR, DNA sequencing to monitor BIR mutagenesis of the expanded ATTCT repeat and flanking DNA. In the same aim, we also investigate the role of the replication stabilizing proteins STN1, COPS2 and Pol η in preventing BIR at ATTCT microsatellites. This was done by siRNA knockdown of the proteins of interest, flow cytometry to monitor changes in flow profile after knockdown and sequencing inverse PCR products to detect mutagenesis signatures. Aim 2: To determine the effect of different patient-derived ATTCT repeats (SCA10 variants) and variations in these repeats' sequences on genome stability. Aim 3: To determine the effect of ATTCT repeats and variations in these repeats' sequences o (open full item for complete abstract)
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    Committee: Michael Leffak Ph.D. (Advisor); Michael Kemp Ph.D. (Committee Member); Thomas Brown Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member); Michael Markey Ph.D. (Committee Member) Subjects: Molecular Biology

  • 5. Kavuri, Naga Swathi Sree Validation of a cell line model for studying XPD protein function in Nucleotide Excision Repair

    Master of Science (MS), Wright State University, 2023, Pharmacology and Toxicology

    The various environmental factors like toxic chemicals, chemotherapeutic agents and UV radiation impact human health by altering physiological functions of the normal living cells. One of the main consequences of exposure to these factors is DNA damage. Although, living system have inherent DNA repair mechanisms such as Nucleotide Excision Repair (NER), which removes DNA lesions from the genome in the form of small, approximately 30-nt-long DNA oligonucleotides. The accumulation of unrepaired DNA adducts as a result of this NER deficiency causes a variety of syndromes, including Xeroderma Pigmentosum (XP), Cockayne Syndrome, Neurological syndrome, and carcinoma of skin. To understand more about NER and to prevent these disorders we need proper experimental/cell line model. Transcription factor II-H (TFIIH) is a ten-subunit protein complex that plays a role in RNA polymerase II (Pol II) transcription initiation and in NER. The excised oligonucleotides released from DNA by NER initially associate with TFIIH and likely specifically with the XPD subunit. To better understand how the excised oligonucleotides bind to and are released from XPD to promote the recycling of TFIIH for new rounds of NER, in this project we characterized an XPD mutant CHO cell line expressing a Flag-tagged human XPD. We found that expression of this protein promoted cellular resistance to UV and other agents that generate DNA adducts repaired by NER and enabled UV photoproduct removal from the genome. However, problems were encountered in detecting the excised oligonucleotide products of NER in these cell lines, and thus various approaches aimed at improving assay conditions. Nonetheless, our results suggest that these cell lines will be useful to characterize XPD function and association with the excised oligonucleotide products of NER.
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    Committee: Michael G. Kemp Ph.D. (Advisor); Yong-jie Xu M.D., Ph.D. (Committee Member); Ravi P. Sahu Ph.D. (Committee Member) Subjects: Pharmacology; Toxicology

  • 6. Chen, Christopher Cellular and Viral Factors Governing DNA-PK Activation During Adenovirus Infection

    Doctor of Philosophy, Miami University, 2022, Microbiology

    The stability and integrity of the genetic information inscribed in DNA molecules are essential to all living organisms and are under constitutive surveillance by sensor proteins that will elicit the DNA damage response (DDR) upon detecting DNA lesions. The presence of input and replicating viral genomes in infected cells may also trigger the cellular DDR, as viral genomes and replication intermediates can have structures that mimic features of damaged DNA. Double-stranded DNA breaks (DSBs) can be catastrophic for cell viability and are predominately repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is canonically regulated by the Mre11-Rad50-Nbs1 (MRN) sensor complex. In contrast, NHEJ is mediated by the DNA-dependent protein kinase (DNA-PK) complex, consisting of the Ku70/Ku86 (Ku) sensor proteins and the DNA-PK catalytic subunit. DSBs induced by chemotherapeutics such as etoposide result in DNA ends with covalently-attached proteins. The resolution of these DSBs requires nucleolytic processing by the MRN complex to remove these proteins and complete NHEJ. The molecular mechanisms governing the collaboration of the MRN and DNA-PK complexes in resolving these lesions are not fully understood. The purpose of this study is to investigate the cellular and viral factors that govern DNA-PK activation during adenovirus (Ad) infection. Ad contains a double-stranded DNA genome with a covalently-attached terminal protein (TP) at each 5' end. Since Ad infection elicits DDRs that can obstruct viral propagation, Ad encodes several proteins from early region 4 (E4) that can limit the function of DDR factors. Both E4-11 and -34 kDa interact with DNA-PK and inhibit some of its activities. Infection with an Ad mutant lacking E4 genes (E4-) results in the concatenation of viral genomes via NHEJ. This process requires the removal of the 5' covalently-attached TP by the MRN complex and DNA-PK for NHEJ-mediated ligation. The data indicates viral (open full item for complete abstract)
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    Committee: Eileen Bridge (Advisor) Subjects: Microbiology

  • 7. Gogusetti, Vivek Shashank Nag Functions of ATR Kinase in Terminally Differentiated Human Epidermal Keratinocyles and in Human Ex-Vivo Skin After Exposure to Ultraviolet B Radiation

    Master of Science (MS), Wright State University, 2021, Pharmacology and Toxicology

    The functions of Ataxia telangiectasia and Rad-3 related protein (ATR) is very much important in a cell, as it is a DNA damage response protein, which plays an important role in cell division, DNA repair and apoptosis. This protein helps in proliferation in the actively DNA dividing normal cells and in cancer cells. The functions of ATR in a proliferating cell are well studied and known to involve regulation of replication fork and cell cycle progression after DNA damage. Whereas, in a non-replicating cell, the functions of ATR are not so well known. In the human body, most of the cells are in a non-replicating state, which do not actively replicate DNA, and include cells in a quiescent, senescent, and terminally differentiated state. What could be the function of ATR in these cells is something that nobody has ever looked at and is important because differentiated cells are routinely exposed to DNA damaging agents. ATR inhibitors are used as combination treatments in DNA damage-based anti-cancer therapies to inhibit pro-survival functions of ATR in cancer cells. Some of the studies show that, inhibition of ATR in non-diving cells would show an opposite effect than in the diving cells in response to DNA Damage caused by UVB. Hence, we have conducted experiments to test if inhibition of ATR would show a pro-survival role in differentiated keratinocytes. DNA damage has been induced using UV-B radiation and ATR is activated in both differentiated N-TERT keratinocytes in vitro and in human skin.
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    Committee: Michael G. Kemp Ph.D. (Advisor); Jeffrey B. Travers Ph.D., M.D. (Committee Member); Yong-Jie Xu Ph.D., M.D. (Committee Member) Subjects: Aging; Atmosphere; Atmospheric Sciences; Biomedical Research; Climate Change; Clinical Psychology; Environmental Science; Environmental Studies; Health Care; Health Education; Health Sciences; Medicine; Molecular Biology; Oncology; Pharmaceuticals; Pharmacology; Pharmacy Sciences; Toxicology

  • 8. Vilfranc, Chrystelle Elucidating the Role of BRUCE in Chronic Liver Disease Pathogenesis

    PhD, University of Cincinnati, 2021, Medicine: Cancer and Cell Biology

    BIR repeat containing ubiquitin-conjugating enzyme (BRUCE) is a giant protein (530kD) that was initially identified as an inhibitor of apoptosis and has also been implicated in processes mouse embryogenesis and efficient DNA damage repair. A previous study implicated BRUCE as a guardian of chromosome integrity and genomic stability and an activator of ATM-dependent DNA double-strand break repair. Still there was no implication of BRUCE in the regulation of the alternative repair kinase ATR, a master regulatory kinase that activates the replication stress response to overcome replication barriers. Replication fork stability during DNA replication is necessary for maintenance of genomic stability and suppression of cancer development in mammals. This work aimed to understand what molecular mechanisms are regulated by BRUCE that can contribute to disease regulation. This work demonstrated new functions for BRUCE in ATR activation in vitro and in liver tumor suppression in vivo. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by the genotoxin Diethylnitrosamine, which can indirectly contribute to replication stress. Liver-specific knockout (LKO) of murine Bruce impaired ATR activation and exacerbated inflammation and liver cancer. In humans, BRUCE downregulation in liver diseases including hepatocellular carcinoma (HCC) specimens and deleterious somatic mutations of the Bruce gene was found in human HCC in the TCGA database. These findings helped to establish a new BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans. I further investigated the role of BRUCE in liver tumor suppression`. Studies have established a pathologic connection between fibrosis and HCC in patients. In mice, DEN exposure alone does not induce robust hepatic fibrosis. By using th (open full item for complete abstract)
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    Committee: Chunying Du Ph.D. (Committee Chair); Paul Andreassen Ph.D. (Committee Member); Tom Cunningham Ph.D. (Committee Member); David Hui Ph.D. (Committee Member); Vladimir Kalinichenko M.D. Ph.D. (Committee Member); Susan Waltz Ph.D. (Committee Member) Subjects: 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. Tikhomirova, Anastasiia Studies of Photoinduced DNA Damage by Phenanthrene Dihydrodioxin and Light-driven Electron Delocalization in Pyridinium Molecules

    Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Photochemical Sciences

    Ever since the discovery of the DNA molecular structure, this molecule became a desirable target for the researchers aiming to develop anticancer drugs and therapies. The main emphasis of this dissertation was placed on the design, development and studies of photochemical properties of a novel photo activated DNA damaging agent. Upon light irradiation of compound the release of highly reactive 9,10-phenanthrenequinone was observed. Studies of phenanthrene dihydrodioxin interaction with DNA demonstrated the intercalative mode of binding and slightly preferential binding affinity to the AT-rich DNA sequences. The synthesized agent induced a partial transition from B to Z form of DNA. The phenanthrene dihydrodioxin compound proved to be an efficient single-strand DNA photocleaver upon visible light irradiation. This further demonstrates the potential of photomasked ortho-quinones as efficient DNA damaging agents. This work also describes an unusual transformation from pyridinium substituted methane to the corresponding gem-diol in the mild conditions in the presence of air. Proposed mechanism of the reaction involves the formation of reactive oxygen species (ROS). ROS are known to be highly toxic to biomolecules, including DNA. It is demonstrated in this work that the production of ROS intermediates in the reaction of the gem-diol formation can lead to DNA damage in the dark. In the continuous interest of our research group, a series of bis(pyridinium) and bis(3-carboxamidepyridinium) alkane salts were synthesized in order to study through-space electron delocalization and formation of dimer radical cation species upon electrochemical and photoreduction. Investigation of photoinduced charge separation is important for the development of artificial photosynthetic systems and molecular electronics. Studies of unsubstituted and meta-substituted pyridines connected by the alkyl linker of different length provided additional information on the efficiency of the pyridi (open full item for complete abstract)
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    Committee: R. Marshall Wilson Prof. (Advisor); Farida Selim Prof. (Other); Pavel Anzenbacher Prof. (Committee Member); Alexander Tarnovsky Prof. (Committee Member) Subjects: Biochemistry; Chemistry; Organic Chemistry; Physical Chemistry

  • 12. 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

  • 13. Kavanaugh, Gina The Role of the Human DEK Oncogene in the Regulation of DNA Damage Response and Repair

    PhD, University of Cincinnati, 2011, Medicine: Cell and Molecular Biology

    Altered chromatin integrity and improper DNA damage repair are commonly associated with the formation and progression of human cancers. Here, we investigate the contribution of the DEK oncogene – elevated in numerous human cancers – to DNA damage repair and chromatin stability. Initially, studies examining the effect of DEK loss demonstrated an accumulation of low levels of DNA damage markers in human cancer cells and xenografts, suggesting a deficiency in DNA damage repair. Importantly, DEK depletion led to alterations in DNA damage kinase response pathways. ATM pathway activation was increased, accounting for the elevated levels of H2AX phosphorylation observed in DEK deficient cells. Alternatively, DNA-PK phosphorylation and kinase activity were reduced with DEK loss. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end-joining (NHEJ), micro-homology mediated end-joining (MMEJ), and homologous recombination (HR) repair when compared to their wild type counterparts. Notably, DEK-depleted Hela cells exhibited reduced levels of histone H2B ubiquitination. Histone modifications play an important role in genome topology and DNA damage repair. Additionally, H2B ubiquitination was recently reported to be required for the recruitment of NHEJ and HR components. We therefore hypothesize that DEK plays a role upstream of the DNA damage response as a regulator of chromatin stability and topology. Future studies concentrating on the contribution of DEK to genome integrity may reveal possible molecular DEK targets for the development of cancer therapeutics.
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    Committee: Susanne Wells PhD (Committee Chair); Paul Andreassen PhD (Committee Member); John Bissler MD (Committee Member); Karl Matlin PhD (Committee Member); Shao-Chun Wang PhD (Committee Member); Yi Zheng PhD (Committee Member) Subjects: Molecular Biology

  • 14. Behbehani, Gregory The BLM Helicase Is Involved in the Repair of DNA Lesions Induced by Diverse Genotoxins

    PhD, University of Cincinnati, 2007, Medicine : Molecular Genetics, Biochemistry, and Microbiology

    Bloom's syndrome (BS) is an autosomal recessive inherited disorder characterized by small size, immunodeficiency, and a striking predisposition to cancer. Cells from persons with BS demonstrate genomic instability, characterized by chromosome breaks, gaps, quadraradials, and a marked elevation in SCE. The gene mutated in BS has been identified and encodes a DNA helicase known as BLM. The mechanism by which BLM deficiency leads to cancer predisposition in persons with BS remains to be elucidated. My work focuses on understanding the role of the BLM helicase in the cellular response to DNA damage induced by diverse genotoxins. Using lymphoblast cell lines isolated from persons with BS, and from age- and sex-matched control individuals, I tested growth capacity in response to several genotoxic agents. I demonstrated BS cell sensitivity to mitomycin C (MMC), hydroxyurea (HU), and a low sensitivity to ionizing radiation (IR). To test sensitivity in vivo, Blm heterozygous mice were treated with large doses of IR and MMC. These experiments demonstrated that heterozygous mice have a slight sensitivity to IR, but surprisingly not to MMC. To confirm that BLM is involved in the response to DNA damage induced by these agents, I demonstrated that a GFP-BLM fusion protein could co-localize with phosphorylated H2AX at sites of DNA damage, and that this co-localization was much greater following DNA damage induced by MMC and HU than damage induced by IR. Lastly, I used flow cytometry to demonstrate that the kinetics of H2AX phosphorylation and de-phosphorylation are largely normal in BS cells, with only a slight delay in cell cycle progression following MMC exposure. I also investigated the formation of foci of H2AX phosphorylation in the breast cancer cell line MCF7 following treatment with the histone deacetylase inhibitors (HDI) trichostatin A (TSA) and sodium butyrate (NaB). I demonstrated that these foci were correlated with double strand DNA breaks as treatment of MCF7 cells (open full item for complete abstract)
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    Committee: Dr. Joanna Groden (Advisor) Subjects: Health Sciences, Oncology

  • 15. 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:

  • 16. Keirsey, Jeremy Phosphorylation regulation of the function, localization and protein interactions of the BLM helicase

    Master of Science, The Ohio State University, 2012, Pathology

    Bloom's syndrome (BS) is a genetic instability disorder resulting from loss of function of the BLM helicase. It confers a marked predisposition to most types of cancer. BS cells are characterized by excessive inter- and intra-chromosomal exchanges as well as chromosomal aberrations highlighting the important role of BLM in maintaining DNA fidelity. BLM unwinds a variety of nucleic acid structures and catalyzes branch migration, single-strand annealing and double Holliday junction dissolution. BLM interacts with proteins involved in DNA damage recognition, signaling and repair. The biochemical activities and protein interactions of BLM are important for various aspects of nucleic acid metabolism including double-strand break repair, DNA replication repair, telomere maintenance and rDNA transcription. BLM undergoes post-translational modification to coordinate its various activities. This study investigates the effect of one of these post-translational modifications, phosphorylation, on BLM function. Helicase assays were performed with in vitro expressed and purified BLM proteins to show that BLM phosphorylation inhibits the specific activity of BLM. Putative BLM phosphorylation sites were identified using bioinformatics prediction programs. Evolutionary conservation, secondary structure and phospho-motif analyses were completed to identify putative BLM phosphorylation sites. Site-directed mutagenesis generated phosphomimetic and alanine replacement constructs for sixteen of twenty-two putative phosphorylation sites for use in cell biology and biochemical screens. Eight phosphorylation sites were identified that may regulate BLM helicase activity in vitro. Phosphorylation of some BLM residues was validated using mass spectrometry. The effect of BLM phosphorylation on the replication fork-regression activity of BLM was assessed to determine whether phosphorylation differentially regulates the function of BLM. These studies were carried out in collaboration with Dr. Da (open full item for complete abstract)
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    Committee: James Waldman Ph.D. (Advisor); Tatiana Oberyszyn Ph.D. (Committee Member); Traci Wilgus Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Genetics; Health Sciences; Molecular Biology; Oncology

  • 17. Fiala, Kevin A kinetic and biochemical approach to understanding the mechanisms of novel DNA polymerases

    Doctor of Philosophy, The Ohio State University, 2007, Biochemistry

    DNA polymerases are the enzymes responsible for the vital task of faithfully duplicating genomes in order to pass on these genetically encoded instructions to their offspring. However, the process of faithfully propagating this information is hindered in all organisms due to endogenous and exogenous agents that damage DNA. While DNA repair mechanisms correct the vast majority of the resulting DNA lesions, unrepaired lesions do persist in the presence of fully functional repair mechanisms. Fortunately cells have evolved a class of promiscuous enzymes known as lesion bypass polymerases that have been shown to bypass DNA lesions that stall the high fidelity replicative DNA polymerases. Here, we have studied two DNA polymerases, human DNA polymerase e and Sulfolobus solfataricus DNA polymerase IV (Dpo4), which are thought to be involved in the previously mentioned cellular processes of DNA repair and DNA lesion bypass respectively. In the process of establishing a minimal kinetic mechanism for the incorporation of a single nucleotide into undamaged DNA catalyzed by human DNA polymerase e, we discovered a novel mechanism in which one of its non-enzymatic N-terminal domains, the Proline-rich domain, dramatically increases the fidelity of the C-terminal DNA polymerase a-like domain by 10- to 100-fold to the level equivalent to that observed with DNA polymerase a, with which it shares 33% sequence identity. Moreover, we have also explored the effects of various structurally distinct DNA substrates on the catalytic efficiency of nucleotide incorporation where we determined the downstream strand and its 5'-phosphate increase the incorporation efficiency by 15- and 11-fold respectively. We have used S. solfataricus Dpo4 as a model Y-family DNA polymerase to elucidate the kinetic mechanism for nucleotide incorporation at both 37 °C and 56 °C, demonstrating that Dpo4 uses an induced-fit mechanism to select and incorporate a correct nucleotide into undamaged DNA independent of re (open full item for complete abstract)
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    Committee: Zucai Suo (Advisor) Subjects: Chemistry, Biochemistry

  • 18. Connelly, Sandra Effects of Ultraviolet Radiation (UVR) Induced DNA Damage and Other Ecological Determinants on cryptosporidium Parvum, Giardia Lamblia, and Daphnia spp. in Freshwater Ecosystems

    Doctor of Philosophy, Miami University, 2007, Zoology

    Freshwater ecosystems are especially susceptible to climatic change, including anthropogenic-induced changes, as they are directly influenced by the atmosphere and terrestrial ecosystems. A major environmental factor that potentially affects every element of an ecosystem, directly or indirectly, is ultraviolet radiation (UVR). UVR has been shown to negatively affect the DNA of aquatic organisms by the same mechanism, formation of photoproducts (cyclobutane pyrimidine dimers; CPDs), as in humans. First, the induction of CPDs by solar UVR was quantified in four aquatic and terrestrial temperate ecosystems. Data show significant variation in CPD formation not only between aquatic and terrestrial ecosystems but also within a single ecosystem and between seasons. Second, there is little quantitative data on UV-induced DNA damage and the effectiveness of DNA repair mechanisms on the damage induced in freshwater invertebrates in the literature. The rate of photoproduct induction (CPDs) and DNA repair (photoenzymatic and nucleotide excision repair) in Daphniafollowing UVR exposures in artificial as well as two natural temperate lake systems was tested. The effect of temperature on the DNA repair rates, and ultimately the organisms' survival, was tested under controlled laboratory conditions following artificial UVB exposure. The results of these studies suggest a significant interaction of UVR and temperature on individual survival and ultimately population dynamics in freshwater systems. Lastly, freshwater human pathogens have negative effects ranging from gastrointestinal distress in otherwise healthy individuals to death in the immunocompromised and elderly. The control of infectious pathogens in water treatment is imperative. The abiotic and biotic environmental stressors of human pathogens are not well understood. Herein, solar radiation and artificial UVB are shown to significantly decrease the infectivity of Cryptosporidium parvum in vitro. The generalist filter feed (open full item for complete abstract)
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    Committee: Craig Williamson (Advisor) Subjects:

  • 19. Mathew, Shomita Investigating the role of DNA damage signaling events in the cellular interference with Adenovirus replication

    Doctor of Philosophy, Miami University, 2007, Microbiology

    Eukaryotic cells possess mechanisms that monitor breaks in genomic DNA and repair them. The effectors of double strand break repair (DSBR) comprise a variety of proteins, including the Mre11/Rad50/Nbs1 (MRN) complex and the mediator of DNA damage checkpoint protein 1 (Mdc1). The Adenovirus (Ad) genome is linear, double stranded DNA that can be a substrate for “repair” by host DSBR proteins that link genomes end-to-end forming concatemers. The virus defends itself against this repair by producing regulatory proteins that interfere with DSBR. Early region 4 (E4) orf3-11kDa protein relocalizes the cellular MRN complex to nuclear track-like structures. The E4-orf6 34kDa/E1b-55kDa complex targets MRN for proteasome mediated degradation. Mutants that lack the E4-11kDa and 34kDa proteins activate the cellular damage response and are severely defective for DNA replication. We have investigated roles for the MRN complex and Mdc1 as sensors and effectors of damage signaling in an Ad-E4 mutant infection, particularly related to the onset of an efficient viral DNA replication. Briefly, we find that the MRN complex regulates the re-localization of Mdc1 early in the infection with an E4 mutant virus, consistent with its role as a sensor of DNA damage. Mdc1 is re-localized in response to viral infection and binds E4 mutant viral DNA, but does not appear to have a role in the regulation of viral DNA replication. The MRN complex, however, is relocalized to E4 mutant viral DNA replication centers in an Nbs1 dependent manner and binds viral DNA. The Nbs1 dependent binding of the complex to E4 mutant viral DNA inhibits the efficient onset of viral DNA replication consistent with the role of the MRN complex as an effector in the damage response and repair pathway. Investigating the ability of host DSBR proteins to interfere with E4 mutant DNA synthesis provides a model for understanding the mechanism by which these proteins sense and respond to the presence of aberrant or damaged DNA.
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    Committee: Eileen Bridge (Advisor) Subjects:

  • 20. Abdallah, Buthina Independent Generation and Investigation of the C3'-deoxy-3'-thymidinyl Radical: A Proposed Intermediate in DNA-LEE Interactions

    Doctor of Philosophy in Medicinal Chemistry (Ph.D.), University of Toledo, 2011, College of Pharmacy

    Ionizing radiation (IR) can damage biological systems by targeting the genetic material of the cells producing lethal DNA lesions. When not repaired these lesions contribute to cell death. Oxidative damage to DNA through IR is partially produced by the secondary particles created along the ionization track. Secondary low-energy electrons (LEEs, 1-30 eV) are the most abundant secondary species produced by IR. It was proposed that LEE can added to DNA constituents resulting in the formation of transient molecular anion intermediates that eventually lead to bond dissociation. It is believed that carbon-centered radicals on the sugar moiety are a major reactive intermediate formed in this process. One of these is the C3'-deoxy-3'-thymidinyl radical (76). Accordingly, the goal of this project is to elucidate the pathways involved in the fate of this radical in DNA to answer mechanistic questions related to DNA damage by LEEs. Site-selective generation of the C3'-deoxy-3'-thymidinyl radical (76) from photolabile precursors should facilitate mechanistic studies as allows generation of one intermediate. The synthesis of α- and β-C3'-deoxy-3'-(selenophenyl)thymidine (73 and 75) as precursors of this radical has been completed and their efficiencies in radical generation have been established. Photolysis of 73 under anaerobic conditions resulted in the formation of the 2',3'-dideoxythymidine (77), 2',3'-didehydro-2',3'-dideoxythymidine (78) and 3',4'-didehydro-2',3'-dideoxythymidine (79) and the isomeric product 75. We believe that their formation is mainly through secondary reactions by the phenylselenyl radical (27). Photolysis experiments in the presence of bis(tributyltin) (80) suggest a disproportionation mechanism. To investigate the fate of the C3'-deoxy-3'-thymidinyl radical in DNA, the modified nucleosides were incorporated into oligonucleotides using "reverse" DNA synthesis. Photolysis of the selenated oligomers under physiological conditions resulted in formation (open full item for complete abstract)
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    Committee: Amanda Bryant-Friedrich PhD (Committee Chair); Marcia McInerney PhD (Committee Member); Viranga Tillekeratne PhD (Committee Member); Wendell Griffith PhD (Committee Member) Subjects: Chemistry