Theses and Dissertations from UMD

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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM

More information is available at Theses and Dissertations at University of Maryland Libraries.

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    THERMOCOCCUS KODAKARENSIS DNA REPLICATION MACHINERY
    (2012) Pan, Miao; Kelman, Zvi; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    DNA replication is the basis for the propagation and evolution of living organisms. It requires the combined efforts of numerous proteins. DNA replication in archaea has been shown to be more similar to eukarya than bacteria. Therefore, we use archaea as a model to study DNA replication. Euryarchaeon is one of the five main branches of archaeon. In this thesis, the replication machinery of the thermophilic euryarchaeon Thermococcus kodakarensis was investigated. In particular, this work focuses on two essential DNA replication proteins, the minichromosome maintenance (MCM) helicase and the processivity factor, proliferating cell nuclear antigen (PCNA). The MCM complex is thought to function as the replicative helicase in archaea and eukaryotes. In most archaea, one MCM homolog assembles to form the active homohexameric complex. Atypically, the genome of T. kodakarensis encodes three MCM homologs, here designated MCM1-3. Although all three MCM exhibit helicase activity, DNA binding and ATPase activities, only MCM3 appears to be essential for cell viability. Taken together with bioinformatics analysis, the results suggest that MCM3 is the replicative helicase in T. kodakarensis. PCNA is a ring shaped protein that encircles duplex DNA and, upon binding to the polymerase and other proteins, tethers them to the DNA. All euryarchaeal genomes, except T. kodakarensis, encode for a single PCNA protein. T. kodakarensis is unique because it contains two genes encoding for PCNA1 and PCNA2. It is shown here that both PCNA proteins stimulate DNA polymerase activity. It was found that PCNA1 is expressed in vivo at high levels in comparison to PCNA2. Furthermore, it was determined that PCNA2 is dispensable for cell viability. Taking together, the data presented herein suggest that T. kodakarensis is similar to other archaeal species studied, requiring only one MCM and one PCNA protein for viability. The results obtained from this work provide essential knowledge about the replication machinery in eukarya.
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    Biochemical characterization of the Minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus
    (2009) Sakakibara, Nozomi; Julin, Douglas; Kelman, Zvi; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    DNA replication requires coordination of numerous proteins to duplicate genetic information in a precise and timely manner. One of the key players in replication is the replicative helicase that unwinds the duplex DNA to provide the single-stranded template for the DNA polymerases. Minichromosome maintenance (MCM) protein is the replicative helicase in archaea. This dissertation focuses on the MCM helicase from the euryarchaeon Methanothermobacter thermautotrophicus (Mth). Archaeal MCM proteins can be divided into two major parts, the N terminal and C terminal domains. The N terminal domain is essential for DNA binding and multimerization, while the C-terminus contains the catalytic domains. The objective of this dissertation is to elucidate the mechanism by which the N terminal domain communicates with the catalytic domain to facilitate helicase activity. To address this question, two approaches were taken. One approach identified conserved residues found in the N terminus and investigated their properties using various biochemical and biophysical methods. By analyzing several proteins with mutations in the conserved residues, a loop that is essential for MCM helicase activity was identified. The study suggests that the loop is involved in coupling the N-terminal DNA binding function and the catalytic activity of the AAA+ domain. Some other conserved residues, however, did not directly affect the MCM helicase activity but showed differences in biochemical properties suggesting that they may play a role in maintaining the structural integrity of the MCM helicase. Another approach determined the differences in thermal stability of the MCM protein in the presence of various cofactors and DNA substrates. The study shows that the protein has two unfolding transitions when ATP and the DNA are present, while non-hydrolyzable ATP results in one transition. This study suggests possible conformational changes arising from decoupling of two domains that occur during the ATP hydrolysis in the presence of DNA. Furthermore, both DNA binding function by the N terminal domain and ATP binding by the catalytic domain are essential for the change.
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    STRUCTURAL AND FUNCTIONAL ANALYSIS OF DNA REPLICATION INITIATION PROTEINS FROM THE ARCHAEON METHANOTHERMOBACTER THERMAUTOTROPHICUS
    (2005-12-01) Kasiviswanathan, Rajesh; Kelman, Zvi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The faithful duplication of the chromosome requires the combined efforts of numerous proteins. Cdc6 and MCM are two such proteins involved in the initiation of DNA replication. The genome of the euryarchaeon Methanothermobacter thermautotrophicus contains one MCM and two Cdc6 homologues (Cdc6-1 and -2). While MCM is the replicative helicase that unwind the duplex DNA to provide single-stranded DNA substrate for the replicative polymerases, the Cdc6 proteins are presumed to function in origin recognition and helicase assembly at the origin. This thesis elucidates the structure, function and regulation of these archaeal initiation proteins. The M. thermautotrophicus MCM helicase is a dumb-bell shaped double hexamer. Each monomer can be divided into two portions. The C-terminal catalytic region contains the ATP binding and hydrolysis sites essential for helicase activity. This thesis concentrates its efforts to determine the functional role of the N-terminal region. Using a variety of biochemical approaches it was found that the N-terminal portion of MCM is involved in hexamer/dodecamer formation. The study also identified two structural features at the N-terminus, the zinc- and the beta-finger motifs, essential for DNA binding, which in turn is essential for helicase activity. In addition, the N-terminal portion of MCM interacts with both Cdc6 proteins. The role of the Cdc6-1 and -2 proteins in origin recognition and helicase loading was also elucidated. The results presented in this thesis show that Cdc6-1 has binding specificity to origin DNA sequences suggesting a role for the protein in origin recognition. While both Cdc6 proteins interact with the MCM helicase, Cdc6-2 exhibited tighter binding compared to Cdc6-1 suggesting a role for Cdc6-2 in helicase loading. Summarizing the observations of this study, a model for the replication initiation process in M. thermautotrophicus has been proposed, outlining separate role for the two Cdc6 proteins, Cdc6-1 in origin recognition and Cdc6-2 in MCM helicase assembly at the origin.