Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-140).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 72-79).

Modulating chromatin structure is an important step in maintaining control over the eukaryotic genome. SWI/SNF, one of the complexes belonging to the growing family of ATP-dependent chromatin remodeling enzymes, is involved in controlling the expression of a number of inducible genes whose proper regulation is vital for metabolism and progression through mitosis. The mechanism by which SWI/SNF modulates chromatin structure at the nucleosome level is an important aspect of this regulation. The work in this dissertation focuses on how the Saccharomyces cerevisiae SWI/SNF complex uses the energy of ATP-hydrolysis to alter DNA-histone contacts in nucleosomes. This has been approached in a two part fashion. First, the three-dimensional structure and subunit composition of SWI/SNF complex has been determined. From this study we have identified a potential region of the SWI/SNF complex that might [be] a site for nucleosomal interaction. Second, functional analysis of the ATPase domain of Swi2p, the catalytic subunit of SWI/SNF, has revealed that a specific conserved motif is involved in coupling ATP hydrolysis to the mechanism of chromatin remodeling. These results provide a potential model for the function of the SWI/SNF chromatin remodeling complex at the nucleosome level.

The bovine papillomavirus type 1 E2 protein is a multifunctional early viral protein with roles in all phases of the cell cycle. E2 is required during G1 as a transcription factor, in S phase to initiate viral replication and during mitosis to tether the viral genome to dividing DNA. The viral genome contains 17 E2 binding sites, the majority of which are concentrated in the long control region (LCR), a regulatory region that is upstream of the viral coding sequence. The role of these binding sites has been explored in vitro using small plasmids and E1 and E2 proteins expressed in bacteria and insect cells. In this study we attempt to examine the placement of E2 on its binding sites during all phases of the cell cycle and in the context of a stably replicating viral system. As part of the examination of the role of E2 during mitosis, we have also examined the role of the cohesin protein Scc1 in viral tethering. Two groups have published disparate reports identifying the cellular protein that binds to the transactivation domain of E2 to stably maintain viral genomes during cell division. Our group has published that it is the DNA helicase ChlR1 that is required for viral tethering, while it has been reported that it is the bromodomain protein Brd4 that is responsible. In this study we contribute to a report that shows that the cellular protein Scc1 binds to the viral genome through a ChlR1 independent mechanism. The cohesin protein binds to BPV-1 E2 at intermittent stages of the cell cycle and may be a factor in viral genome tethering. This interaction may also be important for regulating viral transcription.

DNA-binding proteins (DBPs) such as repair proteins, DNA polymerases, re- combinases, transcription factors, etc. manifest diverse stochastic behaviours dependent on physiological conditions inside the cell. Now that multiple independent in vitro studies have extensively characterised different aspects of the biochemistry of DBPs, computational and mathematical tools that would be able to integrate this information into a coherent framework are in huge demand, especially when attempting a transition to in vivo characterisation of these systems. ChIP-Seq is the method commonly used to study DBPs in vivo. This method generates high resolution sequencing data { population scale readout of the activity of DBPs on the DNA. The mathematical tools available for the analysis of this type of data are at the moment very restrictive in their ability to extract mechanistic and quantitative details on the activity of DBPs. The main trouble that researchers experience when analysing such population scale sequencing data is effectively disentangling complexity in these data, since the observed output often combines diverse outcomes of multiple unsynchronised processes reflecting biomolecular variability. Although being a static snapshot ChIP-Seq can be effectively utilised as a readout for the dynamics of DBPs in vivo. This thesis features a new approach to ChIP-Seq analysis { namely accessing the concealed details of the dynamic behaviour of DBPs on DNA using probabilistic modelling, statistical inference and numerical optimisation. In order to achieve this I propose to integrate previously acquired assumptions about the behaviour of DBPs into a Markov- Chain model which would allow to take into account their intrinsic stochasticity. By incorporating this model into a statistical model of data acquisition, the experimentally observed output can be simulated and then compared to in vivo data to reverse engineer the stochastic activity of DBPs on the DNA. Conventional tools normally employ simple empirical models where the parameters have no link with the mechanistic reality of the process under scrutiny. This thesis marks the transition from qualitative analysis to mechanistic modelling in an attempt to make the most of the high resolution sequencing data. It is also worth noting that from a computer science point of view DBPs are of great interest since they are able to perform stochastic computation on DNA by responding in a probabilistic manner to the patterns encoded in the DNA. The theoretical framework proposed here allows to quantitatively characterise complex responses of these molecular machines to the sequence features.

Chromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA. The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partnerbinding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding. Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery.

Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control. Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease. The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain. We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys. We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia. Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease. In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21886号 / エネ博第387号 / 新制||エネ||75(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB) are among the most lethal forms of DNA damage and can occur as a result of ionizing radiation (IR), radiomimetic agents, endogenous DNA-damaging agents, etc. If left unrepaired DSB’s can cause cell death, chromosome translocation and carcinogenesis. In humans, DSB are repaired predominantly by the non-homologous end joining (NHEJ) pathway. Ku, a heterodimer consisting of Ku70 and Ku80, functions in the recognition step of this pathway through binding DNA termini. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to create the full DNA-PK heterotrimer. Formation of DNA-PK results in autophosphorylation as well as phosphorylation of downstream proteins of the NHEJ pathway. Previous work shows that the extreme C-terminus of Ku80 stimulates the kinase activity of DNA-PKcs, and Ku DNA binding is regulated as a function of redox via stimulation of a conformational change when oxidized resulting in a decrease in DNA binding activity. To further understand these methods of regulation of Ku and DNA-PK, a pair of mutants has been constructed; one consisting of full length Ku70 and truncated Ku80 (Ku70/80ΔC) lacking 182 C-terminal amino acids. The removal of these amino acids was shown to have little to no effect on the proteins expression, stability or DNA binding, as determined by SDS-PAGE, western blot analysis and electrophoretic mobility shift assay (EMSA). When oxidized Ku70/80ΔC showed a decrease in DNA binding similar to that seen in wild type, however when re-reduced the mutant did not recover to the same extent as wild type. A second mutant was constructed, containing amino acids 590-732 of Ku80 (Ku80CTR), to further understand the mechanism by which Ku80 C-terminus interacts with the rest of the Ku heterodimer. Possible protein-protein interactions were evaluated by Ni-NTA affinity, gel filtration chromatography, fluorescence polarization and two forms of protein-protein cross-linking. Ni-NTA agarose affinity, and gel filtration chromatography failed to reveal an interaction in the presence or absence of DNA. However, photo-induced cross-linking of unmodified proteins (PICUP) as well as EDC cross-linking demonstrated an interaction which was not affected by DNA. The work presented here demonstrates that the interaction between Ku80CTR and Ku is rather weak, but it does exist and plays a relatively large role in the NHEJ pathway.