Doctor of Philosophy, The Ohio State University, 2020, Chemistry

Chromatin is a protein-DNA complex. It compacts the eukaryotic genome by a factor of ~10,000 times to confine the genome in the cell nuclei. Additionally, chromatin regulates the function of the genome, such as replication, transcription, and repair, by limiting the access of the regulatory proteins to the genome. Nucleosome is the building block of chromatin with a histone octamer protein complex at the core, and ~147 bp of DNA wrapped around the histone core. Despite a nearly 50-year modern scientific investigation, there is still a lot to understand about chromatin structure and function. There are several challenges in studying chromatin. The molecular weight of a single nucleosome is ~200 kDa, and it contains intrinsically disordered domains protruding out of the nucleosome. Crystal structures of mononucleosome and tetranucleosomes were determined before. However, histone tails escape high-resolution structure determination by X-ray crystallography. The dynamic characteristics of chromatin cannot be captured by crystallography, as well. Therefore, several other biophysical and biochemical approaches have been employed to study the structure and function of chromatin, namely Cryo-EM and single-molecule experiments. NMR is another powerful tool to study the structure and dynamics of biological samples. It allows probing the dynamics of histone tails directly at a near-atomic resolution, which is not readily feasible by any other technique. In this dissertation, a biochemical technic was used to probe the accessibility of histone tails in nucleosome arrays. These arrays of nucleosomes are the model systems for in-vitro studies of chromatin. Additionally, solution and solid-state NMR methods were employed to study the dynamics of histone tails and the structure of the histone core region in both nucleosomes and nucleosome arrays. The results from the biochemical kinetic studies indicate that this method is a promising tool to determine the accessibility (open full item for complete abstract)

Committee: Christopher Jaroniec Professor (Advisor); Heather Allen Professor (Committee Member); Ross Dalbey Professor (Committee Member) Subjects: Biochemistry; Chemistry

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2009, Biological Sciences

High mobility group box-1 protein (HMGB1) increases the in vitro binding affinity of estrogen receptor (ER) to the various estrogen response elements (EREs) such as consensus ERE (cERE), tandem cEREs, consensus half-site ERE (cHERE), tandem cHEREs, and variant spacer cEREn, n= 0-4bp), while decreasing the binding specificity. To test if this in vitro binding characteristic translates to the functional activity in the cell, firefly luciferase reporter vectors were constructed with different EREs noted above at 5- to the TATA box upstream of the luciferase gene. Transient transfection of these constructs was performed in estrogen treated, ER negative human osteosarcoma cell lines, U2OS, along with the co-transfection of mammalian expression vectors pERα and pHMGB1. The in vitro binding activity of HMGB1 was correlated with the functional activity in cells, but there was no linear correlation between in vitro and in vivo. HMGB1 stimulated the transcriptional synergy for tandem cEREs and cHEREs which is reflected in its influence on in vitro binding co-operativity. cEREns with variant spacers, to which the HMGB1 had increased the ER binding affinity very strongly, did not produce a strong transcriptional activity even though it enhanced the activity. HMGB1 knock down experiments with siRNA in determining the influence of HMGB1 on estrogen (E2) responsive gene expression clearly showed that HMGB1 is involved in E2 responsive gene expression.ER binds strongly to cERE on free DNA but does not bind (KD >200nM) to cERE in nucleosomal DNA. However, the presence of HMGB1 greatly facilitates ER binding (KD~50nM) to cERE in the nucleosome. To determine the influence of HMGB1 on nucleosome structure which could have facilitated enhanced binding of ER to cERE in nucleosomal DNA, characteristic of the nucleosome were studied after treating it with increasing levels of HMGB1 (up to 1600nM). The structure of the nucleosome is markedly altered by HMGB1 interaction, with the population (open full item for complete abstract)

Committee: Dr. William Scovell PhD (Advisor); Dr. Carmen Fioravanti PhD (Committee Co-Chair); Dr. Carol Heckman PhD (Committee Member); Dr. George Bullerjahn PhD (Committee Member); Dr. Bruno Ullrich PhD (Committee Member) Subjects: Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2024, Physics

DNA is compacted in the eukaryotic cell nucleus into chromatin by wrapping ~147 bp of DNA at a time around protein histone octamer cores composed of 2 each of histones H2A, H2B, H3, and H4. Repeats of this DNA-protein complex, which is referred to as the nucleosome, form higher order chromatin structure, and it plays a large role in gene transcription regulation by controlling access to DNA. Multiple mechanisms exist to overcome the nucleosomal barrier to transcription. Each of the histones in the nucleosome can have post-translational modifications (PTMs) made to specific residues in the histone as a way to directly affect the structure of the nucleosome, or signal for other cellular machinery to open access to the DNA. These modifications can affect each other across the nucleosomes in a cell. Often the deposition of one type of modification will affect the state of another type of histone modification on the surrounding nucleosomes. This network of histone PTMs contributes to the epigenetic landscape of the cell directing cellular machinery in the nucleus to perform its various functions, and has been referred to as the histone code mechanism. In addition, there exist proteins capable of depositing these modifications on histones, binding specifically to the modifications, or removing them from histones, and these proteins are considered the writers, readers, and erasers of the histone code. A certain number of these proteins do not exclusively perform only one of these functions, and will contribute to the network of histone PTMs in a more complex manner by reading and targeting one PTM type while being responsible for depositing or removing another PTM type for instance. The category of histone PTM related proteins is vast, and so this thesis will focus on characterizing histone PTM reader type proteins and their interactions with modified histone chromatin in vitro. Work in this thesis showed that PTM readers are capable of a wide variety o (open full item for complete abstract)

Committee: Michael Poirier (Advisor); Ezekiel Johnston-Halperin (Committee Member); Comert Kural (Committee Member); Ralf Bundschuh (Committee Member) Subjects: Biophysics; Physics

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

Doctor of Philosophy, The Ohio State University, 2019, Chemistry

Solid-state NMR (SSNMR) spectroscopy is an incredibly powerful tool for studying the structure and dynamics of biomolecules and large macromolecular complexes. SSNMR has no inherent size restriction and is useful in studying non-crystalline protein such as membrane-bound protein or amyloid fibrils, DNA-protein complexes, and large protein assemblies such as the HIV-capsid. Meanwhile, techniques such as solution-state NMR and x-ray crystallography are strictly limited by molecular size and sample condition, and thus cannot study large biomolecules or insoluble protein aggregates, respectively. The first protein at the center of this study is the human prion protein (PrP) and its formation into an insoluble amyloid fibril. The formation of this fibril leads to a deposition of an insoluble plaque on the central nervous system which leads to the development of the prion protein disease, known as Gerstmann-Straussler-Scheinker (GSS) disease. The second system-of-interest is the nucleosome core particle (NCP) which is a DNA-protein complex and is the building-block of chromatin and chromosomes. An individual NCP is composed of dsDNA wrapped around an octamer of histone protein, mimicking the natural phenomena of DNA storage and compaction in eukaryotic cells. SSNMR is uniquely qualified in studying these biological systems in depth to characterize the amyloid fibril propagation and disease-causing mechanism of prion protein, and to explore nucleic acid base-pairing behavior at DNA-protein interfaces and the important interactions therein. Dynamic nuclear polarization (DNP) is a hyperpolarization technique for NMR spectroscopy which dramatically increases the overall sensitivity of these experiments. In DNP-SSNMR, hyperpolarization is achieved by applying microwave irradiation to free electrons, often in the form of stable-radicals, within a static magnetic field and inducing a polarization transfer to neighboring nuclear spins, especially spin-½ protons. These elevated (open full item for complete abstract)

Committee: Christopher Jaroniec (Advisor); Rafael Bruschweiler (Committee Member); Joshua Goldberger (Committee Member); Marcos Sotomayor (Committee Member) Subjects: Chemistry

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

Scaffolded DNA origami uses DNA as building blocks to create 3D nanostructures. Dynamic 3D DNA origami nanostructures have strong potential in biophysical experiments and applications. This includes measuring biomolecular forces, particularly on the nanoscale. Current biophysical methods lack the ability and/or specificity to measure on such a scale, in addition to being challenging and expensive. Dynamic DNA origami can address such a problem by providing a malleable material to accommodate shape and function on the 10-100nm length scale. The major goal of this work is to leverage DNA origami to develop tools to study biophysical properties of the biomolecular complexes. Nucleosomes, consisting of genomic DNA wrapped around a protein core, assemble into higher orders of chromatin structure to compact DNA. Tools to probe site-specific chromatin structure and dynamics at the 10-100nm length scale (relevant for gene regulation) and to apply tensile or compressive forces at targeted sites could greatly improve insight into how chromatin structural dynamics regulate DNA accessibility and processing. We designed, constructed, and implemented a nanocaliper via DNA origami. Our nanocalipers are hinge-like joints that consist of two 70nm rigid arms, each made up of bundled DNA helices, connected along one edge by single stranded DNA. For proof-of-concept, we first studied a single nucleosome by binding the two nucleosomal DNA ends to the ends of nanocaliper arms. Here, the caliper angle reports the nucleosome end-to-end distance. Moreover, the caliper angle is sensitive to nucleosome stability as a function of NaCl concentration. We demonstrated the nanocaliper can detect nucleosome conformational changes via transcription activator Gal4-VP16 binding. The caliper also significantly increases the probability of Gal4-VP16 occupancy by applying a tension to partially unwrap the nucleosome. This suggests that our DNA nanocalipers can report biologically relevant conformat (open full item for complete abstract)

Committee: Carlos Castro (Advisor); Michael Poirier (Committee Member); Marcos Sotomayor (Committee Member); Jonathan Song (Committee Member) Subjects: Biochemistry; Biomechanics; Biophysics; Mechanical Engineering

Doctor of Philosophy, The Ohio State University, 2018, Biomedical Sciences

HIV is a worldwide pandemic that remains incurable. Recent statistics show that in the United States alone, ~15 per 100,000 people were newly infected with HIV-1 in one year. The barrier to a cure is a reservoir of cells with viral DNA stably integrated into their genome, yet are not killed by the immune system. The integration step of the retroviral life cycle is crucial in reservoir formation. Viral DNA integration is catalyzed by protein integrase (IN). We study HIV-1 IN and prototype foamy virus (PFV) IN. PFV IN is used to model for HIV-1 integration, as HIV-1 IN inhibitors also block PFV IN activity. This implies that the two proteins have similar catalytic mechanisms. However, we have found differences between PFV IN and HIV-1 IN function. We determined that PFV IN could utilize calcium for strand transfer, unlike HIV-1 IN. Additionally, though HIV-1 IN has been reported to rapidly commit to its target DNA, PFV IN does not commit within an hour. There are likely differences in searching and target capture mechanisms between the two INs. A benefit to using PFV IN is that it can be readily assembled with oligomers that mimic viral cDNA ends to form an intasome complex. The PFV intasome contains a tetramer of PFV IN and two oligomer DNAs. We found in vitro that these intasomes aggregate at 37°C. Full-length intasomes aggregate more than those containing truncated PFV IN outer subunits, particularly deleting the carboxyl terminal domain (CTD). Aggregation can be prevented by using high non-physiological salt concentrations or with addition of small molecule protocatechuic acid (PCA). This finding is useful for future experiments that require longer lifetimes of PFV intasomes. Integration into chromatin is not well understood. Chromatin is comprised of basic units called nucleosomes. Our goal is to understand how IN chooses its site when integrating into nucleosomes. We altered either nucleosomes or PFV IN to understand how changes impact integration acti (open full item for complete abstract)

Committee: Kristine Yoder (Advisor); Michael Freitas (Committee Member); Jesse Kwiek (Committee Member); Li Wu (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Virology

Doctor of Philosophy, The Ohio State University, 2016, Physics

All the DNA in a cell's nucleus is packaged into a material called chromatin consisting of DNA and DNA-associated proteins. The basic unit of chromatin is the nucleosome which consists of ~147 bases of DNA wrapped around a protein core composed of two copies each of histones H2A, H2B, H3, and H4. DNA wrapped into a nucleosome is inaccessible to most DNA processing machinery. This machinery needs access to the DNA to perform processes such as transcription, replication, and repair. The cell uses many mechanisms to modulate this protection including nucleosome unwrapping, sliding, remodeling, disassembly, and chemical modification. This work used transcription factors along with fluorescent methods to probe the accessibility of DNA near the ends of the nucleosome. The DNA in this entry-exit region can spontaneously unwrap to provide transient access to DNA binding factors such as transcription factors. We studied the effect of histone post-translational modifications on the accessibility of DNA in the entry-exit region. We found that the phosphorylation at H3Y41ph increased DNA accessibility in vitro by 3-fold, which is similar in size to the effect of a previously studied modification H3K56ac. Since both of these modifications are associated with active genes and could occur together, we studied nucleosomes with both modifications and they showed a 17±5-fold increase in accessibility. This indicates that the cell could use multiple modifications in the entry-exit region to adjust the accessibility of nucleosomal DNA by over an order of magnitude. We also studied the effects of the removal of one H2A/H2B dimer. These partially formed nucleosomes, called hexasomes, are a component of chromatin that has been largely unstudied. Our results have shown that hexasomes have DNA accessibility similar to that of fully-formed nucleosomes on the side with the remaining H2A/H2B dimer. However, on the side without the dimer, our results showed the DNA remains permanently unwr (open full item for complete abstract)

Committee: Michael Poirier (Advisor); Ralf Bundschuh (Committee Member); Comert Kural (Committee Member); Louis DiMauro (Committee Member) Subjects: Biochemistry; Biophysics; Physics

Doctor of Philosophy, The Ohio State University, 2016, Physics

The eukaryotic genome is organized into a structural polymer called chromatin. Ultimately, all access to genetic information is regulated by chromatin including access required for DNA replication, transcription, and repair. The basic repeating unit of chromatin is the nucleosome which is comprised of ~147 bp of DNA tightly wrapped around a protein histone octamer core. The histone octamer is made up of eight proteins: two each of histones H2A, H2B, H3, and H4. Many mechanisms exist to regulate access to DNA but one of pivotal importance is the creation of unique nucleosomes through i) integration of histone variants and ii) deposition of post translational modifications (PTMs). These modifications help comprise the epigenome of a cell. Classically, the two mechanisms by which they function have been through a direct regulation of nucleosome dynamics, or through third party proteins which are able to recognize the variants or PTMs and facilitate work. The library of potential PTMs therefore forms a sort of histone code which regulates access to DNA. This thesis investigates the intersection of these mechanisms to determine whether the act of recognizing epigenetic information alters DNA accessibility. The primary method used to determine changes in DNA accessibility is though observing the effective binding affinity of a transcription factor to its target site buried within a recombinantly prepared nucleosome which has been modified to carry a PTM and to report on its wrapping state. We find different regulation depending both on the PTM we investigate and the specific PTM-binding protein. We first investigate the H3K36me3-binding protein PHF1 and find that while the PTM it recognizes, H3K36me3, does not alter DNA accessibility, the binding of its recognition domain and N-terminal domain can illicit a change of DNA accessibilty of 8 ± 2-fold. This means that 8 times less DNA binding protein is required to occupy its target site if the nucleosome is bound by PH (open full item for complete abstract)

Committee: Michael Poirier PhD (Advisor); Ralf Bundschuh PhD (Committee Member); Comert Kural PhD (Committee Member); Fengyuan Yang PhD (Committee Member); Michael Barton PhD (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Molecular Biology; Physics

Doctor of Philosophy, The Ohio State University, 2015, Biophysics

Nucleosome is the most basic structural and functional unit of the eukaryotic genome. In each nucleosome, 147 base pairs of genomic DNA is wrapped 1.6 turns around a histone octamer protein core. The structural dynamics of nucleosome plays important role in the regulation of many gene-related cellular processes such as transcription activation, DNA replication and repair. This dissertation work focuses on the interplay between nucleosomal DNA unwrapping and transcription factor (TF) accessing the target site within the nucleosome. TFs are important regulatory proteins that bind their target sites on the genomic DNA to activate or repress transcription. The access of TF is blocked if a nucleosome is positioned on the target site, which occurs often in the genome. The nucleosomal DNA at the entry/exit region of the nucleosome undergoes rapid spontaneous unwrapping and rewrapping from the histone core under thermal fluctuation, allowing transient exposure of the target site for binding. We employed Fluorescence Resonance Energy Transfer (FRET) method to probe the conformation changes of nucleosome unwrapping and detect TF binding to the nucleosome-protected sites. Single-molecule Total-internal-reflection fluorescence (smTIRF) microscopy was used to track this dynamic process in real time. It was revealed in this study that nucleosomes regulate TF binding not only by reducing its association rate with limited site exposure, but also by accelerating the dissociation of TF from a nucleosomal site. The results showed that the overall TF dissociation rate is enhanced by a factor of 2-3 orders of magnitude. This enhanced dissociation appears to be a new general mechanism by which nucleosomes could regulate TF occupancy in the genome. Further investigations showed that this enhanced dissociation rate remains largely unchanged as the binding site is moved across a long stretch of DNA in the entry/exit region of nucleosome, and the TF occupancy at these sites is mainly regulat (open full item for complete abstract)

Committee: Michael Poirier Dr. (Advisor); Ralf Bundschuh Dr. (Committee Member); Carlos Castro Dr. (Committee Member); Richard Swenson Dr. (Committee Member) Subjects: Biochemistry; Biology; Biomechanics; Biophysics; Molecular Biology; Physics

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

Eukaryotic DNA is organized into a structural polymer called chromatin which ultimately controls important DNA processing functions such as transcription, DNA repair and DNA replication. The fundamental unit of chromatin is the nucleosome which is made up of about 146 base pairs wrapped around a histone core. The histone core contains 2 copies each of the histones H2A, H2B, H3 and H4. Long strings of nucleosomes compact into higher order structures which are not well known, but play a pivotal role in DNA accessibility. There are many factors that affect higher order structure and compaction of chromatin including inter and intra nucleosome interactions, incorporation of linker histones (H1), and post translational modifications. This dissertation includes a detailed study of some of these mechanisms. The first study looks at the H3 N-terminal tail which is long, unstructured and heavily modified in vivo . Using Electron Paramagnetic Resonance and site directed spin labeling; we were able to observe the dynamics of the H3 tail within compacted 17-mer nucleosome arrays. We find that these tails maintain their mobility as the arrays compact and self-associate despite previous studies that suggested these tails make inter- and intra-nucleosome contacts during compaction. We conclude that these contacts are transient and permit the tails to maintain mobility and accessibility. The second study looks at how H1 affects transcription. Using Fluorescence Resonance Energy Transfer and Protein Induce Fluorescence Enhancement, we observed the binding of H1 to the nucleosome, while also being able to detect the binding of a transcription factor (TF) within the entry-exit region of the nucleosome. We find that H1 represses TF binding by a factor of three for unmodified nucleosomes. These results indicate that H1 does not block butonly suppresses DNA accessability and that H1 exchange is not the only process by which DNA can be accessed. We also observed H1 interacting wit (open full item for complete abstract)

Committee: Michael Poirier PhD (Advisor); Ralf Bundschuh PhD (Committee Member); R Sooryakumar PhD (Committee Member); Richard Furnstahl PhD (Committee Member) Subjects: Biophysics; Physics

Doctor of Philosophy, The Ohio State University, 2012, Physics

The DNA in eukaryotic cells is organized into a tightly-regulated structural polymer called chromatin that ultimately controls crucial functions of the genome, including gene expression, DNA synthesis, and repair. The basic unit of chromatin is the nucleosome in which 147 base pairs of DNA wraps 1.7-times around eight "core" histone proteins (two copies each of H2A, H2B, H3, H4). Repeats of this structural unit have been shown to fold into higher order structures, which play a central role in controlling DNA accessibility for transcription regulation. However, at the individual nucleosome level, DNA-histone interactions that wrap DNA into the nucleosome also control DNA accessibility. A significant number of factors have been shown to regulate nucleosome accessibility, including variants and post-translational chemical modifications of to the core histone proteins, chromatin remodeling complexes that reposition and disassemble nucleosomes, and histone chaperones that deposit or remove histones. Ultimately, these chromatin regulatory factors must physically alter nucleosomes to change DNA accessibility to transcription, replication, and DNA repair machinery. This work encompasses a detailed study of the integral relationship between histone post-translational modifications (PTMs) and DNA accessibility. There are more than 100 reported PTMs throughout the nucleosome, many of which serve as binding sites for chromatin regulatory proteins. However, a subset of these PTMs are buried beneath the DNA-histone interface and are seemingly inaccessible to regulatory proteins. Given that nucleosomes in vivo typically possess multiple PTMs, until recently it has been difficult to determine the precise function of PTMs residing in the DNA-histone interface. Using fully-synthetic and semi-synthetic protein ligation strategies in conjunction with the Ottesen Lab, we have engineered and incorporated histones bearing precise PTMs into nucleosomes for biophysical characterization. Add (open full item for complete abstract)

Committee: Michael Poirier PhD (Advisor); Jennifer Ottesen PhD (Committee Member); Ralf Bundschuh PhD (Committee Member); Comert Kural PhD (Committee Member); Said Sif PhD (Committee Member) Subjects: Biophysics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2011, Physics

We investigate the use of physical modeling to extract mechanistic details from quantitative biological data, with a focus on the physical properties of nucleic acids. It is well understood that DNA stores genetic information, RNA acts as a carrier of this information, and that both must interact with a wide array of protein complexes in order to perform these functions. However, the physical mechanisms by which these interactions occur are much less clear. For example, Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We test this model for three sequences at temperatures from 23C to 65C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. There are also many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV and the RecA DNA repair protein in bacteria. The presence of such proteins can strongly alter the secondary structure of the nucleic acid molecules. Therefore, accurate modeling of the interaction between single-stranded nucleic acids and such proteins is essential to fully understanding many biological processes. We develop a model for predicting nucleic acid secondary structure in the presence of single stranded binding proteins, and implement it as an extension of the Vienna RNA Package. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid, and FRET distributions for (open full item for complete abstract)

Committee: Ralf Bundschuh (Advisor); Michael Poirier (Advisor); Mohit Randeria (Committee Member); Samir Mathur (Committee Member); David Symer (Committee Member) Subjects: Biophysics

Doctor of Philosophy, The Ohio State University, 2010, Biophysics

The human MutS homologues (MSH), hMSH2 and hMSH6, forms a heterodimer (hMSH2-hMSH6) that plays a central role in mismatch repair (MMR). hMSH2-hMSH6 is required for the recognition of mismatched nucleotides and insertion/deletion loops (IDLs) generated by misincorporation during DNA replication. Mutations in either of the hMSH2 or hMSH6 genes result in elevated spontaneous mutation rate and susceptibility to the common cancer predisposition syndrome, Lynch Syndrome or hereditary non-polyposis colorectal cancer (LS/HNPCC). Mismatches that are recognized by hMSH2-hMSH6 arise in vivo within chromosomes that are a complex mixture of DNA and protein (chromatin). A fundamental unit of chromatin is the nucleosome which consists of ~147 bp of DNA wrapped twice around a histone octamer containing two H2A-H2B dimers and an H3-H4 tetramer. The biophysical/biochemical effect of chromatin on MMR is unknown. Moreover, little is known about the effect of more than a 100 post-translational modifications (PTMs) that may decorate the human histones during MMR processes. This dissertation discusses chromatin and MMR. Chapter 1 serves as an introduction to MMR and chromatin. Chapter 2 provides the thesis rationale. Chapter 3 involves analysis of a mismatched DNA substrate containing a single well-defined nucleosome. We demonstrate that hMSH2-hMSH6 can catalyze the disassembly of a nucleosome adjacent to a mismatch. In addition, we have constructed nucleosomes containing acetylations of the histone H3 dyad residues K115 and K122 by a semi-synthetic intein-based strategy. We find that hMSH2-hMSH6 nucleosome disassembly is considerably enhanced when nucleosomes contain H3(K115, K122) acetylation modifications. Moreover, lysine to a glutamine substitution mutation of histone H3(K56), used to mimic the lysine acetylation, also enhances nucleosome disassembly. Disassembly of the nucleosome requires ATP binding by hMSH2-hMSH6. In addition, nucleosome disassembly is blocked by LacI/LacO pl (open full item for complete abstract)

Committee: Richard Fishel PhD (Advisor); Charles Brooks PhD (Committee Member); Mark Foster PhD (Committee Member); Joanna Groden PhD (Committee Member) Subjects: Biophysics