Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
Browse
5 results
Search Results
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 Resistance to Ionizing Radiation and Oxidative Stress in Halobacterium salinarum NRC-1(2009) Robinson, Courtney Kathryn; Dinman, Jonathan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oxidative stress results from environmental challenges that cause unchecked production of reactive oxygen species (ROS). We analyzed the cellular damage and stress response of the extremophile Halobacterium salinarum NRC-1 exposed to chemical oxidants and to ionizing radiation (IR). In contrast to IR, cellular damage from H2O2 and superoxide suggested that cell death resulted from interference with major metabolic pathways rather than generalized oxidative lesions. We found that essential ROS scavenging enzymes were not necessary for H. salinarum NRC-1 survival to IR. Protection assays using enzyme-free cellular extracts from H. salinarum NRC-1 demonstrated high level of protection for protein activity but not for DNA integrity against IR. Biochemical analysis of the extracts underlined an essential role in ROS scavenging for specific nucleosides and MnPO4 complexes. These studies contributed novel findings on the critical role played by non-enzymatic systems in IR resistance in H. salinarum NRC-1.Item DNA mismatch repair and response to oxidative stress in the extremely halophilic archaeon Halobacterium sp. strain NRC-1(2008-08-14) Busch, Courtney Rae; DiRuggiero, Jocelyne; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Halobacterium is an extremely halophilic archaeon that has homologs of the key proteins, MutS and MutL used in DNA mismatch repair in both Bacteria and Eukarya. To determine whether Halobacterium has a functional mismatch repair system, we calculated the spontaneous mutation rate and determined the spectrum of mutation in Halobacterium using fluctuation tests targeting genes of the UMP biosynthesis pathway and we performed a sequence analysis of the mutated genes. We found that Halobacterium has a low incidence of mutation indicating that some form of DNA repair is taking place, however the mutational spectrum in the Archaea is different from that seen in Bacteria and Eukarya suggesting differences between the archaeal, bacterial, and eukaryal repair systems. To test if the MutS and MutL homologs in Halobacterium are essential for the low incidence of mutation, we used in-frame targeted gene deletion and characterized the mutant phenotypes. We found no phenotypic differences between the mutant strains and the background strain indicating that the MutS and MutL protein homologs found in Halobacterium are not essential for maintaining the low incidence of mutation. Since much of the replication and repair processes in Halobacterium are similar to that of Eukarya, deciphering how MMR occurs in the Archaea could lead to a new understanding of pathway interactions based on the recruitment of repair enzymes from both bacterial and eukaryal counterparts. In addition, we elucidated the oxidative stress response in Halobacterium to hydrogen peroxide and paraquat using a whole genome transcriptional array, in-frame targeted gene deletion, and survival analysis of mutant phenotypes. We showed an overall effort of the cells to scavenge reactive oxygen species and repair damages to the DNA, which has also been seen in response to gamma irradiation. From the mutant analyses, we were able to deduce that Sod1 and PerA proteins played an essential role in removing oxidative stress in Halobacterium. Deciphering the stress response to hydrogen peroxide and paraquat in an extreme halophile that lives in an environment subject to long periods of desiccation can further our understanding of the DNA repair and protection systems to oxidative stress in general.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.