<p>Preserving genome stability is critical to ensure the faithful transmission of intact genetic material through each cell division. One of the key components of this preservation is maintaining low levels of mutagenesis. Most mutations arise during replication of the genome, either as polymerase errors made when copying an undamaged DNA template or during the bypass of DNA lesions. Many different DNA repair proteins act both prior to and during replication to prevent the occurrence of these mutations. Although the mechanisms by which mutations occur and the various repair proteins that act to suppress mutagenesis are conserved throughout all species, they are best characterized in the yeast <italic>Saccharomyces cerevisiae</italic>. In this work, we have used this model system to study two types of spontaneous mutagenesis: oxidative mutagenesis and frameshift mutagenesis. In the first part of this work, we have examined mutagenesis that arises due to one of the most common oxidative lesions in the cell, 7,8-dihydro-8-oxoguanine or GO. When present during replication, these GO lesions generate characteristic transversion events that are accurately repaired by the mismatch repair pathway. We provide the first evidence that a second pathway involving the translesion synthesis polymerase Pol&eta acts independently of the mismatch repair pathway to suppress GO-associated mutagenesis. We have also examined how differences in replication timing during S phase contribute to variations in the rate of these mutations across the genome. In the second part of this work, we have examined how spontaneous frameshift mutations are generated during replication. While most frameshift mutations occur in regions of repetitive DNA, we have designed a system to examine frameshifts that occur in very short repeats (< 4 nucleotides) and noniterated sequences. We have examined the patterns of frameshifts at these sites and how the mismatch repair pathway acts to suppress these mutations. Together, the experiments presented here provide further insight into the different mechanisms that suppress and/or influence rates of oxidative mutagenesis and describe a system in which we have begun to characterize how frameshift mutations are generated at very short repeats and non-repetitive DNA.</p> / Dissertation

Human T-cell lymphotropic virus type I (HTLV-I) is a retrovirus that is the causative agent of the fatal disease adult T-cell leukemia (ATL). HTLV-I silently infects over twenty million people worldwide; up to ten percent of these will develop ATL in their lifetime. There are currently no effective treatments for this disease. HTLV-I expresses its genome as polypeptides that must be processed in order to produce infectious virions. Like other retroviruses, HTLV-I encodes an aspartic acid protease to process these polypeptides into mature form. Because the protease is essential in the virus life cycle, it is an attractive target for the treatment of HTLV-I-induced ATL. The present work examines the structure and function of HTLV-I protease. A theoretical structure of the protease is presented, and the function of the C-terminal extension is considered. In order to determine which residues are involved in binding substrate, two experiments were performed: first, several residues were mutated to the corresponding residues in HIV-1 protease to determine whether HTLV-I protease can be made to process an HIV-1 protease substrate; second, an alanine scan was performed to knock out individual residues to assess their importance in binding substrate. This work builds knowledge of the structure and function of HTLV-I protease. By understanding which residues play a role in binding substrate and by developing a clearer picture of the structure of the protease, it will be possible to develop specific inhibitors for HTLV-I protease.

Nuclear protein KLIP1 cooperates with myeloid leukemia factor 1 (MLF1) to inhibit the programmed cell death resulting in tumor formation. It also inhibits the activity of thymidine kinase promoter of Kaposi¡¦s sarcoma-associated Herpes Virus. KLIP1 functions as a centromere protein, hence acquires its name as CENP-U or CENP-50, to regulate the separation of sister-chromatids during mitosis. These results indicate that KLIP1 plays important roles in regulation of transcription and cell cycle. In this study, six potential SUMO modification sites, K33, K63, K126, K127, K185 and K210, were identified bioinformatically using SUMOplot. Many reports address that SUMO modification alters the transcriptional activity, protein-protein interaction, the subcellular localization and stability of its target protein. Recent data suggest that SUMO is required for centromere binding protein to mediate proper mitotic spindle attachment to the kinetochore, and previous research suggest that there has a SUMO-interaction motif (SIM) in KLIP1 protein sequence. To reveal the interaction between KLIP1 and SUMO-1, and study its effects on KLIP1 function, we co-express GFP-KLIP1 and His-tagged SUMO-1 in HEK 293 cells. After affinity purification of SUMOylated proteins from transfected cells using nickel conjugated beads and subsequent western blotted with anti-GFP. The results indicated the interaction between KLIP1 and SUMO-1 in co-transfected cells. Our confocal microscopy imaging also found colocalization of GFP-KLIP1 with RFP-SUMO-1 nuclear foci. In addition, we failed to detect the interaction between SUMO-1 and mutant KLIP1-M6 ,whose six potential SUMO modified lysine residues were mutated to arginine. Furthermore, we found a distinct nuclear localization of GFP-KLIP1-M6 as compared to the image of wildtype GFP-KLIP1, which show a significant higher frequency of colocalization with RFP-SUMO-1 foci. Taken together, our data suggest the interaction between KLIP1 and SUMO-1 may be related to these six potential lysine residues, which upon mutation blocks its colocalization with SUMO-1 in nuclear foci. The biological significance of their interaction are awaits for further investigation.

Abstract: TSG101 is a tumor susceptibility gene exhibits multiple biological function, including the regulation of cell progression, intracellular protein sorting and membrane trafficking, and transcription activity of nuclear recptor such as estrogen recptor. TSG101 contains an UBC domain which is homologous to that in ubiquitin conjugating E2 enzyme. However, it lacks an essential cysteine residue, which is essential for catalytic activity. Cellular protein ubiquitination serves as a signal for protein degradation or sorting into multivesicular body. UBC domain of TSG101 was proved to contain amino acid residues that are important for its interaction with ubquitin (residues V43, N46, D46 and F88) and PTAP sequence found in the late domain of HIV gag protein (residues Y63, M95, V141). SUMO is an ubquitin-like modifier which can modify cellular protein harbors £ZKXE amino acid sequence, thereby change its subcellular localization and biological activities. TSG101 protein contains K98, K243, K264 and K269 residues that localize in potential SUMO modification site. Our preliminary data indicated that TSG101 colocalize with SUMO in nucleus. It is interesting to know whether TSG101 is sumoylated, and its functional significance. In this thesis, a series of site-directed mutageneic mutant HA and GFP-tagged expression plasmids which contain mutation of the above mentioned functional related amino acid residues were constructed for future TSG101 functional studies.

Thesis (M.S.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2010. Title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ56223.

DNA replication demands extraordinary specificity and efficiency of catalysis from a DNA polymerase. Previous studies on several DNA polymerases suggested that a rate-limiting conformational change preceding chemistry accounts for the high specificity following the induced fit mechanism. However, the identity of this rate-limiting conformational change and how it contributes to the fidelity is still under debate. An important study of T7 DNA polymerase performed by Tsai and Johnson using a conformationally sensitive fluorophore (CSF) characterized a conformational change directly and presented a new paradigm for nucleotide selectivity. This thesis describes work to further characterize the underlying molecular basis regulating the conformational change by a combination of site-directed mutagenesis, transient kinetics and crystallography. One flexible segment (gly-ala-gly) within the fingers domain was mutated to (ala-alaala). The kinetic analysis on this mutant showed that the mutations decreased the forward rate of the conformational change reported by the fluorophore about 1200-fold but there was no significant change on the reverse rate. The data suggested that the movement of the fingers domain is not a rigid body motion but may be complex due to the movements of various helices within the fingers domain. Quantification of the kinetics of incorporation of correct and incorrect base pairs showed the decrease of fidelity mainly was from the decreased forward rate during correct nucleotide incorporation. The roles of three active site residues, K522, H506, and R518, which form polar interactions with [alpha]-,[beat]- and [gamma]-phosphates of the incoming nucleotide respectively, in conformational change and catalysis were also characterized. All the mutants showed a slower conformational change than the wild type enzyme. After this conformational change, there was a rate limiting step with a rate comparable to kpol measured by quench-flow experiments. Correct nucleotide binding caused an increase in fluorescence, suggesting that the conformational change of the fingers domain delivers incoming nucleotide to a misaligned status even for a correct nucleotide with each of the mutants. The data suggested that active site residues play important roles in maintaining a fast conformational change and an accurate alignment of the active site during correct nucleotide incorporation. Yellow crystals of CSF-labeled T7 DNA polymerase with DNA and correct nucleotide (closed complex), incorrect nucleotide (misaligned complex) or no nucleotide (open complex) were grown to good size and diffracted to 3 Å during X-ray data collection. The structures of these complexes are still under refinement. / text

No description available.

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

The effects of a newly mapped DNA repair-defective mutant, uvsI, on mutagen sensitivities and mutation were investigated. Results showed that uvsI differs for most of the investigated properties from other uvs mutants of A. nidulans which are known to belong to three different epistatic groups, "UvsF", "UvsC", and "UvsB". Most of these mutants are sterile and many of them alter mitotic recombination frequencies, while uvsI exhibits normal levels of meiotic and mitotic recombination. In addition, uvsI strains are not more sensitive than wild type to MMS (methyl methanesulfonate) to which all other uvs strains are sensitive. However, the uvsI mutant was found to be very sensitive to the killing effects of UV light and the chemical mutagen, 4-NQO (4-nitro-quinoline-N-oxide). In line with the distinct phenotype of uvsI, no epistatic interactions were found for this mutant with any members of the established three epistatic groups. The effects of uvsI on mutagenesis are highly specific and dependent on the mutational test systems. In the uvsI mutant, two types of forward mutation were not affected, but spontaneous and UV-induced reversion frequencies of choA1 and pabaA1 were significantly reduced. Specific effects were further demonstrated in reversion tests of various sC alleles originally isolated as selenate resistant mutants by treatment with EMS (ethyl methanesulfonate), which leads mainly to G:C to A:T transitions. After EMS treatment uvsI mutants showed highly reduced reversion frequencies for all these sC alleles (except one) compared to $uvs sp+$ strains. These results suggest that the uvsI mutation may be defective in AT to GC transition mutagenesis, while increasing transversion(s) from A:T base pairs. In contrast, uvsI affected the frequencies of spontaneous and UV-induced reversions for these sC alleles in a variety of ways. Thus, uvsI may well represent a fourth functional and epistatic group of DNA repair and possibly be involved in a minor mutagenic DN