Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

The human genome relies on DNA repair proteins and the telomere to maintain genome stability. Genome instability is recognized as a hallmark of cancer, as is limitless replicative capacity. Cancer cells require telomere maintenance to enable this uncontrolled growth. Most often telomerase is activated, although a subset of human cancers depend on recombination-based mechanisms known as Alternative Lengthening of Telomeres (ALT). ALT depends invariably on recombination and its associated DNA repair proteins to extend telomeres. This study tested the hypothesis that the requirement for those requisite recombination proteins include other types of DNA repair proteins. These functions were tested in ALT cell lines using C-circle abundance as a marker of ALT. The requirement for homologous recombination proteins and other DNA repair proteins varied between ALT cell lines compared. Several proteins essential for homologous recombination were dispensable for C-circle production in some ALT cell lines, while proteins grouped into excision DNA repair processes were required for C-circle production. The MSH2 mismatch repair protein was required for telomere recombination by intertelomeric exchange. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between human cancer cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways. Further studies of all DNA repair pathways in ALT will likely lead to a better understanding of ALT mechanisms and ultimately better ALT-targeted therapeutics.

Committee: Jeffrey Parvin MD PhD (Advisor); Joanna Groden PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2001, Medicine : Molecular Genetics, Biochemistry and Microbiology

Multi-celled organisms are mosaic in nature because somatic mutations occur in individual cells of the body. Little is known about the frequency of mutation in most cell types of mammals. To make this phenomenon more amenable to study, transgenic mice carrying a histochemically detectable reporter gene were created, allowing for individual cells with a particular frameshift mutation to be detected. In order to enhance the chances of detecting spontaneous mutation events, the transgene was modified to contain a hypermutable region of 11 G:C basepairs. The mutation frequencies in mouse brain, heart, kidney and liver were quantified, yielding information about how often -1 frameshift mutations occur in the wildtype mouse genome. Although no particular tissue had consistently more mutation events than another from the same mouse, these frequencies tend to be, on average, 5- to 7-fold higher in brain and heart as compared to liver and kidney (i.e. ~20 events vs. ~2 events per million cells). This suggests that the tissues each have different methods for coping with mononucleotide runs. The DNA mismatch repair (MMR) system is responsible for recognizing and repairing DNA basepair mismatches that can result from recombination, chemical modification or DNA replication errors. The frameshift reporter transgene was crossed into mice lacking a functional Pms2 mismatch repair gene, the products of which are known to be involved in the repair of insertion/deletion events. Mutant frequency increased dramatically in this background, and was estimated to be at least 1000-fold greater than the respective wildtype tissues in brain, heart and kidney. However, frequency increased only 10-fold in liver. These data corroborate the evidence that Pms2 plays a significant role in the maintenance of genome stability at repetitive sequences, and suggest that Pms2 operates in a tissue-specific manner. To investigate this possibility, the effect of Mlh1-deficiency was also studied. Mlh1 is know (open full item for complete abstract)

Committee: James Stringer (Advisor) Subjects:

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, 2007, College of Graduate Studies

Mismatch repair (MMR) corrects mispaired bases and insertion/deletion loops and has long been regarded as a post-replicative DNA repair process. MMR proteins also are involved in DNA damage surveillance, damage-induced cell cycle arrest, apoptosis, and other DNA repair pathways. Biochemical mechanisms of MMR within human cells are not well understood and the involvement of the MMR pathway in correcting DNA mispairs at oncogenic hotspots is unknown. Additionally, cell cycle regulation of MMR proteins has not been well defined. MMR protein expression, binding activity, and repair activity were quantified using proliferating cells and discrete cell cycle populations separated by centrifugal elutriation. Specifically, the quantitative functioning of the MMR system at an oncogenic hotspot sequence located at H-ras codon12 during different cell cycle phases was investigated. We demonstrate that hMutSa is able to recognize both codon 12G:T and 12G:A mismatches, but binding activity and accurate nick-directed repair is greater for 12G:T. Further, MMR proficient cells are able to more accurately repair mismatches at codon 12G:T versus 12G:A, while MMR deficient cells are unable to efficiently correct either mismatch. Additionally, the cell regulates MMR proteins and their activity in a cell cycle dependent manner, which may contribute to efficient MMR at hotspots. Nuclear MMR protein levels increase as the cell progresses from G1 to S phase and remain elevated during G2 phase. Importantly, this work is the first to describe the sustained increase of nuclear levels of all four major mismatch proteins: hMSH2, hMSH6, hPMS2, and hMLH1 during S to G2 cell cycle transition. Interestingly, hMutSa binding and nick-directed repair activity is measureable in G1, increases during S phase, but contrary to protein expression this activity decreases in G2. Further, nick-directed MMR activity is greater in assays using a replication-competent plasmid versus a replication-incompetent plasmi (open full item for complete abstract)

Committee: Kandace Williams, PhD (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2021, Physics

Here I develop computationally efficient quantitative models to describe the behavior of DNA-based systems. DNA is of fundamental biological importance, and its physical properties have been harnessed for technological applications. My work involves each of these aspects of DNA function, and thus provides broad insight into this important biomolecule. First, I examine how DNA mismaches are repaired in the cell. Protein complexes involved in DNA mismatch repair appear to diffuse along dsDNA in order to locate a hemimethylated incision site via a dissociative mechanism. I study the probability that these complexes locate a given target site via a semi-analytic, Monte Carlo calculation that tracks the association and dissociation of the complexes. I compare such probabilities to those obtained using a non-dissociative diffusive scan, and determine that for experimentally observed diffusion constants, search distances, and search durations in vitro, both search mechanisms are highly efficient for a majority of hemimethylated site distances. I then examine the space of physically realistic diffusion constants, hemimethylated site distances, and association lifetimes and determine the regions in which dissociative searching is more or less efficient than non-dissociative searching. I conclude that the dissociative search mechanism is advantageous in the majority of the physically realistic parameter space, suggesting that the dissociative search mechanism confers an evolutionary advantage. I then turn to synthetic DNA structures, initially focusing on a composite DNA nano-device. In particular, manipulation of temperature can be used to actuate DNA origami nano-hinges containing gold nanoparticles. I develop a physical model of this system that uses partition function analysis of the interaction between the nano-hinge and nanoparticle to predict the probability that the nano-hinge is open at a given temperature. The model agrees well with experimental data and pre (open full item for complete abstract)

Committee: Ralf Bundschuh PhD (Advisor); Carlos Castro PhD (Committee Member); Michael Poirier PhD (Committee Member); Hirata Christopher PhD (Committee Member) Subjects: Biophysics; Nanotechnology; Physics; Polymers; Theoretical Physics

Bachelor of Science, Walsh University, 2019, Honors

The human protein PMS1 is a protein that functions in DNA mismatch repair. PMS1 is part of the High Motility Group Protein family (HMG proteins). Using homology modeling in YASARA, a 3-dimensional structure of the PMS1 protein was produced and the structure was verified as realistic using molecular dynamics. Using Evolutionary Analysis in an online program called ConSurf identified the high and low conservative regions of the PMS1 protein. Sequences with high conservation scores indicate important structural and functional aspects of the proteins. Using the GNOMAD database, human variants of the protein were found with a focus placed on those that caused missense, loss of function and frameshift mutations which can be found in the 3-dimensional structure. Where these proteins are found will be at the sites of phenotypical consequences from mutation. Using the Human Protein Atlas, PMS1 was found in all cells. However, it is most common in cells with a high reproductivity rate, like cells in the digestive tract. Malfunctioning of PMS1 leads to genome instability and more frequent mutations, which can cause genetic defects or cancers. The Catalogue of Somatic Mutation in Cancer (COSMIC) was used to identify cancer related mutations of PMS1, such as colorectal cancer. It is hoped that this sequence to structure to function to phenotype approach will contribute to the future of genomic medicine.

Committee: Thomas Freeland B.S., M.S. Ph.D (Advisor); Adam Underwood B.S., M.S., Ph.D (Other) Subjects: Biology

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

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of the repair pathways they are involved in via their tolerance/intolerance of nicks containing mismatched base pairs. This is well illustrated by mammalian short-patch base excision repair (BER), where delay in mismatch ligation catalyzed by the DNA ligase III/XRCC1 complex is expected to provide opportunities for nick editing by the 3' to 5' exonuclease activity of mammalian AP endonuclease (APE). Though a large body of qualitative and quantitative data suggests that DNA ligases from a broad spectrum of organisms are intolerant of non-Watson–Crick base pairs at the 3'–OH terminus of a nick, the recent analysis of the African swine fever virus (ASFV)-encoded DNA ligase demonstrated that there are exceptions to this rule. The ASFV DNA ligase is fairly tolerant of numerous 3' mismatched base pairing schemes and it has been suggested that by efficiently competing with the nick-editing activity of ASFV APE, the ASFV ligase could contribute to the sequence diversity known to exist between different isolates of the virus. Although early studies by T. Lindahl and his colleagues suggested that human DNA ligase I and the ligase III/XRCC1 complex are relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex, which is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway, has not been studied in this regard. During the repair of DSBs generated by chemical/physical damage, as well as the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, error-tolerant DNA ligation would appear to be advantageous. Kinetic characterization of the ligase IV/XRCC4 complex was undertaken in order to examine whether this complex could contribute a mismatched nick ligation activity to NHEJ. (open full item for complete abstract)

Committee: Ming-Daw Tsai (Advisor) Subjects: Chemistry, Biochemistry

PhD, University of Cincinnati, 2012, Arts and Sciences: Biological Sciences

Hyperthermophilic archaea grow optimally at temperatures that accelerate DNA damage which raises important questions about how these organisms maintain genetic fidelity and genome stability. Archaea in general have reshaped our understanding of the different adaptations of cellular life and their uniqueness justifies their classification into a separate domain of life. The aim of the thesis research presented in this document was to investigate genetic fidelity and genome stability in hyperthermophilic archaea. The approach involved developing genetic assays based on conventional bacterial and eukaryal model systems as well as novel approaches to probe fundamental mechanisms of genome stability at the molecular level. An important component of genetic fidelity, DNA mismatch repair, was investigated in Sulfolobus acidocaldarius. Sulfolobus acidocaldarius was found to repair mismatches formed during homologous recombination (HR), which provides the first in vivo evidence for mismatch repair in hyperthermophilic archaea. However, the events seen in S. acidocaldarius were highly localized, involving individual or short patches of mismatches seen within long tracts of mismatched DNAs, and thus differed from that resulting from conventional mismatch repair. This process contributed to the unique properties of HR in S. acidocaldarius compared to known bacterial and eukaryotic counterparts. Evidence for genome stability and genetic fidelity in S. acidocaldarius was obtained by sequencing and comparing whole genomes of three natural isolates from local populations separated by large distances (~8200 km). Only 40 polymorphisms were found across all three strains, which suggest a combination of efficient global dispersal and (novel) genetic mechanisms that limit replication errors and chromosomal rearrangements. DNA exchange via conjugation between S. acidocaldarius cells can contribute to this process. Preliminary studies of the size(s) of DNA fragment(s) transferred and dire (open full item for complete abstract)

Committee: Dennis Grogan PhD (Committee Chair); Edward Merino PhD (Committee Member); Brian Kinkle PhD (Committee Member); Charlotte Paquin PhD (Committee Member); Katherine Tepperman-Elder PhD (Committee Member) Subjects: Microbiology

PhD, University of Cincinnati, 2003, Medicine : Molecular Genetics, Biochemistry and Microbiology

Bloom's syndrome (BS) is a rare autosomal recessive disorder that greatly predisposes affected individuals to cancer. Such individuals also are small in size, sensitive to the sun, have immune dysfunction and gross genomic instability. The cytogenetics of BS cells have been extensively studied and have shown increased levels of homologous recombination, quadriradial formations, telomeric associations and chromosome breakage. The gene responsible for BS has been positionally cloned and and encodes a RecQ helicase family member with strand displacement activity that is dependent on ATP and Mg2+. In order to have a greater understanding of BLM helicase function in the cell in regards to DNA replication, recombination and repair, we identified protein-partners of BLM. The C-terminus of BLM identified the DNA mismatch repair protein MLH1 from a yeast two-hybrid screen. In vitro and in vivo immunoprecipitations confirmed the interaction between these two proteins. Using an in vitro mismatch repair assay, BS cell extracts were tested for their ability to correct a single nucleotide mismatch. The BS cell extracts were able to remove the single nucleotide mismatch from the plasmid DNA, demonstrating that the BLM-MLH1 interaction is not necessary to correct a single nucleotide mismatch substrates. Helicase assays then were performed which demonstrated that MLH1 or the mutL heterodimer modulates the enzymatic activity of BLM by stimulating BLM's strand displacement activity on the double-overhang (DO) substrate. Finally, we performed experiments with the supF20 mutagenesis system and demonstrated that extracts from BS cells are unable to utilize micro-homology elements within the supF20 gene to restore supF function following the induction of a double strand break (DSB). Additional experiments with the pUC18 mutagenesis system demonstrate that although the efficiency and fidelity of DSB repair by BS extracts are comparable to those of normal extracts when ligatable ends are pr (open full item for complete abstract)

Committee: Dr. Joanna Groden (Advisor) Subjects: Chemistry, Biochemistry

Bachelor of Arts, Miami University, 2008, College of Arts and Sciences - Zoology

Accumulation of genomic mutations in hematopoietic and mesenchymal progenitor cells can lead to such diseases as stem cell derived leukemias and lymphomas and sporadic tumors of the gastrointestinal organs. One source of mutation accretion is the dysfunction of DNA Mismatch Repair (MMR), whereby failure to correct specific replication errors may cause the fixation of inappropriate genomic sequences. Microsatellites are repetitive regions of DNA that are highly vulnerable to mutation, so microsatellite instability (MSI) can serve as a marker for MMR dysfunction. We hypothesize that the frequency of MSI in both human mesenchymal stem cells (hMSCs) and human hematopoietic stem cells (hHSCs) increases with age. We have isolated DNA from 12 hMSC colony forming units (CFU) and 6 hHSC CFU of the same patient (ages 30-92). Utilizing PCR with fluorescent primer pairs, five microsatellite loci were amplified for 24 samples from each CFU. The microsatellites were then visualized using polyacrylamide gel electrophoresis and a Typhoon 9200 phosphoimager. A sample was scored positive for MSI if the banding appeared deviant from normal. Each CFU sample was then characterized as MSI-Stable, MSI-Low, or MSI-High. Both hMSCs and hHSCs demonstrated an increase in MSI frequency with age, with a significant increase among the hHSCs that I studied when combined with a larger pool of data of other research (p=0.0034). This pattern suggests an increasing level of MMR dysfunction. The comparison between MSI frequency in hMSCs and hHSCs suggests that the level of accumulation of genomic instability is similar between cell types. This assessment identifies aging hMSCs and hHSCs as a risk factor for downstream tumorigenesis and bone degradation. These cells should be further analyzed as a target for the understanding and treatment of bone and muscle related disorders, but the field of stem cell regenerative medicine should be cautious of the risks of MMR failure.

Committee: Michael Robinson (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy (PhD), University of Toledo, 2012, College of Medicine

Interstrand crosslinks (ICLs) formed by cisplatin are unique lesions formed by the drug. They covalently crosslink both strands of DNA. Cisplatin induced cell death primarily occurs through the formation of various lesions on DNA. Hence, the persistence of the adducts is crucial for its cytotoxicity. DNA repair systems which ensure the integrity of the genome are mainly responsible for the development of resistance to the drug, due to their role in removing the damage on the DNA created by the drug. Repair of ICLs is thought to involve multiple repair pathways. Emerging evidence suggests that there is a replication dependent pathway which depends on stalling of replication forks, and a replication independent mechanism. The exact mechanistic details of cisplatin ICL repair remain poorly understood. Information from studies addressing the repair of ICLs formed by other agents cannot be directly applied to understand cisplatin ICL repair. Each lesion produces unique distortions, and as a result activates different DNA repair pathways. Using cell extracts and purified proteins, we propose a specific pathway for the processing of flanking DNA adjacent to cisplatin ICLs. This mechanism involves the action of two pathways, Base excision repair (BER) and Mismatch repair (MMR) which is activated due to conversion of cytosine adjacent to the cisplatin ICL. We demonstrate that proteins involved in BER –Uracil DNA glycosylase, Apurinic endonuclease, and DNA Polymerase β directly process this uracil. Finally, we showed that action of these BER proteins leads to the activation of MMR downstream of BER. We propose a novel mechanism in which this common mechanistic pathway acts adjacent to cisplatin ICL, but does not influence repair of damage caused by other crosslinking agents. It influences the overall rate of repair by interfering with the repair processes that act to resolve the crosslink.

Committee: Stephan Patrick PhD (Committee Chair); John David Dignam PhD (Committee Member); Ivana De la Serna PhD (Committee Member); Kandace Williams PhD (Committee Member); James Willey PhD (Committee Member) Subjects: Biochemistry

Doctor of Philosophy in Biomedical Sciences (Ph.D.), University of Toledo, 2009, College of Medicine

The Mismatch repair (MMR) system maintains genomic stability byrepairing DNA mismatches and insertion-deletion loops (IDLs) resulting from replication and recombination errors. Defective MMR can lead to hereditary non-polyposis colorectal cancer (HNPCC) and sporadic forms of cancer. In human cells, mismatches are recognized and bound by a heterodimer, hMSH2-hMSH6 (hMutSα). A second heterodimer, hMLH1-hPMS2 (hMutLα) interacts with hMutSα and is thought to act as a mediator for downstream repair proteins. An additional role for MMR pathway is to trigger cell cycle arrest and apoptosis upon recognition and binding of MutSα to specific DNA lesions such as O6 methyldeoxyguanine (O6-meG). Limited information is available regarding the cellular regulation of these proteins. Within this report, we demonstrate that hMSH6, but not hMSH2, undergoes phosphorylation within cells. Phosphorylation of hMSH6 is enhanced by addition of TPA, a PKC activator. Alternatively, UCN-01, a PKC and Chk1/Chk2 kinase inhibitor, decreases hMSH6 phosphorylation and mismatchbinding activity of hMutSα to both G:T and O6 -meG:T-containing DNA. We show that phosphorylated hMSH6 is higher in concentration in the presence of a G:T mismatch, as compared to an O6 -meG:T lesion. However, the total quantity of hMutSα bound to O6 -meG:T–containing DNA is higher than that bound to G:T-containing DNA. We also demonstrate that MMR proficient cells treated with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) undergo cell cycle arrest after one complete cell cycle. When these cells are co-treated with UCN-01 the G2/M arrest is abrogated and the cells rapidly die. This abrogation of arrest is not due to Chk1 kinase inhibition but rather through Cdc2 activation by increased Tyr15 dephosphorylation. Taken together, we hypothesize that both phosphorylation and total concentration of hMutSα are involved in the signaling of either DNA mismatch repair or damage recognition activities. We also hypothe (open full item for complete abstract)

Committee: Kandace Williams Ph.D. (Committee Chair); Stephan Patrick Ph.D. (Committee Member); William Maltese Ph.D. (Committee Member); John David Dignam Ph.D. (Committee Member); Venkatesha Basrur Ph.D. (Committee Member) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2013, Pathology

All organisms undergo complex changes over time. These changes, associated with an increasing incidence of disease and decreased functional capacity, are collectively referred to as aging. To understand this process, to prolong functional capacity, and to prevent the expensive and debilitating diseases associated with increased age is an ongoing focus of scientific study. In the introduction chapter of this dissertation, I present a philosophical argument suggesting aging and age related diseases are a result of intraorganismal genetic change, as a result of failed DNA damage repair, in the adult stem cell (ASC) population. The hematopoietic stem/progenitor cell (HPC) is a well characterized example of ASCs. In second and third chapters I show evidence of age related accumulated genetic instability and epigenetic loss of the DNA mismatch repair (MMR) protein MLH1 expression in the HSCs obtained from healthy normal donors. Based on this observation ASCs cannot, as previously thought, be truly immune to the effects of the aging process, nor is it likely these cells possess infinite expansion potential. In the last chapter of this manuscript, I highlight the impact of these findings and suggest a model of HPC aging which implies age related dysfunction of HSCs is the result of acquired genetic and epigenetic changes incurred over a lifetime.

Committee: Stanton Gerson MD (Advisor); Alan Tartakoff PhD (Committee Chair); Nancy Oleinick PhD (Committee Member); Vincent Monnier MD (Committee Member); Clive Hamlin PhD (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Cellular Biology; Genetics; Gerontology; Health; Molecular Biology