Theses and Dissertations from UMD
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Item ARCHAEAL DNA REPLICATION PROTEINS: MEMBERS AND FUNCTIONS(2013) Li, Zhuo; Kelman, Zvi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The mechanism of DNA replication in archaea, the third domain of life, has been studied for more than two decades using biochemical, structural and bioinformatic approaches. Historically, many of the proteins that participate in archaeal replication were identified via similarity to enzymes needed for DNA replication in bacteria and eukarya. This study uses a different approach to identify new factors that may be involved in replication. Genetic tools developed for the thermophilic archaeon Thermococcus kodakarensis were used to identify new replication factors that could not be recognized through in silico methods. First, a network of proteins that may participate in replication was identified using in vivo tagging of known replication enzymes. Following affinity purification the proteins that co-purified with the tagged enzymes were identified using mass spectrometry. This study describes the identification of a number of new putative replication factors. Next, the biochemical properties of two proteins identified in the screen were characterized. One, the product of gene TK1525, was identified via its interaction with the GINS complex. This protein was predicted to be an archaeal homologue of the bacterial RecJ nuclease. It was found that the protein is a processive, manganese-dependent, single strand DNA-specific exonuclease. The protein was designated GAN for GINS-associated nuclease. GAN forms a complex with GINS and also interacts with the archaeal-specific DNA polymerase D in vivo. Subsequent bioinformatic analysis suggested that GAN may be the archaeal homologue of the eukaryotic Cdc45 protein. The second protein characterized is the product of TK0808. This protein was identified via its interactions with proliferating cell nuclear antigen (PCNA). The protein, upon binding to PCNA, inhibits PCNA-dependent activities. The protein was therefore designated TIP for Thermococcales inhibitor of PCNA. While most proteins that interact with PCNA do so via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA, TIP neither contains the canonical PIP motif nor interacts with PCNA via the IDCL. These findings suggest a new mechanism for PCNA binding and suggest a new mechanism to regulate PCNA-dependent activities.Item 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.Item 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.Item 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.Item PURIFICATION AND CHARACTERIZATION OF THE RECD PROTEIN-HOMOLOGUE FROM DEINOCOCCUS RADIODURANS(2004-12-06) Wang, Jianlei; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In many gram-negative bacteria, RecBCD enzyme is found to be responsible for double strand DNA break repair through homologous recombination. The AddAB enzyme, a RecBCD analog, is found in some gram-positive bacteria and functions in a similar way as RecBCD. A few bacteria appear to lack both RecBCD and AddAB enzymes entirely. One such organism is the bacterium Deinococcus radiodurans. This remarkable organism is able to survive in the presence of very high levels of radiation or DNA-damaging chemicals, levels that would overwhelm the DNA repair capacity of most other organisms. Interestingly, the D. radiodurans genome does have an open reading frame that would encode a protein that is homologous to the E. coli RecD protein. The amino acid sequence of this D. radiodurans RecD-like protein suggests that it is a helicase and therefore could function in some aspect of DNA repair, as does its E. coli homologue. However, the RecD protein of D. radiodurans must serve a different and novel function compared to the E. coli RecD protein. The D. radiodurans RecD protein can be expressed at high levels in E. coli and is readily purified by chromatography on a nickel column followed by single-stranded DNA-cellulose. The purified protein exhibits DNA-dependent ATPase and DNA helicase activities. The helicase activity requires at least a 10 nucleotide single strand overhang at the 5'-end of the double strand DNA substrate to start unwinding. The helicase assay shows that D. radiodurans RecD-like protein unwinds dsDNA substrates catalytically, but with low processivity, even with the help of single strand binding proteins (SSB) from either E. coli or D. radiodurans. These results show that D. radiodurans RecD-like protein is a DNA helicase that moves with 5'-3' polarity on single-stranded DNA. The E. coli RecD protein was shown recently to unwind dsDNA with the same 5'-3' polarity. The low processivity of the D. radiodurans RecD-like protein suggests that it may function in a complex with other proteins. The identity of these proteins is not known. We have also generated insertion mutations that are likely to disrupt all of the recD gene copies in the D. radiodurans genome after multiple generations growing in media with antibiotics. The in vivo effects of the insertion mutation, such as the growth curve and the sensitivity to UV radiation and DNA damaging chemicals, were studied.