Chemistry & Biochemistry Theses and Dissertations
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Item BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THREE DNA REPAIR ENZYMES IN DEINOCOCCUS RADIODURANS(2009) Cao, Zheng; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram positive bacterium Deinococcus radiodurans is able to withstand acute doses of gamma rays that can cause hundreds of double-strand breaks per genome. In proposed double-stand break repair pathways, however, some important enzymes, such as helicases and nucleases in the initiation step, have not been clearly identified yet. Interestingly, the common bacterial helicase/nuclease complex RecBCD or AddAB, which functions to produce a 3' ssDNA tail in double-strand break repair initiation step in other bacteria, is not found in D. radiodurans. As part of efforts to identify helicases involved in double-strand break repair, the D. radiodurans HelIV (encoded by locus DR1572, the helD gene) was characterized with both in vivo and in vitro methods. The helD gene is predicted to encode a helicase superfamily I protein. The helD mutant is moderately sensitive to methyl methanesulfonate and hydrogen peroxide but it is not sensitive to gamma rays, UV and mitomycin C. In biochemical assays, the full length HelIV exhibited DNA unwinding activity with a 5'-3' polarity whereas the truncated HelIV without N-terminal region had no detectable helicase activity. RecJ is the exonuclease in the RecF pathway, which is suggested to function at the initiation step in DSB repair in the absence of RecBCD. In the in vivo study, the D. radiodurans recJ gene (encoded by locus DR1126) cannot be completely removed from the chromosome, indicating the essential role of RecJ in cell growth. The heterozygous mutant displayed growth defect and higher sensitivity to gamma rays, hydrogen peroxide and UV compared to wild type D. radiodurans, suggesting an important role in DNA repair. The RecJ expressed in E. coli system was insoluble but can be purified via denaturation-refolding, and the refolded RecJ showed 5'-3' exonuclease activity. D. radiodurans has no RecB and RecC proteins, but it has a homologue of the RecD protein. We tested whether the D. radiodurans RecD protein could form a complex or make transient interactions with other proteins to perform more complicated functions. The RecD conjugated protein affinity column was used to attempt to identify cellular binding partners.Item Characterization of TATA Box Binding Protein Interaction with Minicircle DNA(2005-10-05) Byun, Jung Shin; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein-induced bending of DNA plays an important role in regulating its transcription, replication, recombination, and packaging into nucleosomes. In particular, many general and gene-specific transcription factors bend DNA. The TATA box-binding protein (TBP) is essential to promoter recognition, and it provides an especially interesting example of dramatic DNA bending. Previous work in our laboratory has shown that TBP bound to strained DNA can induce negative supercoiling, proposed to be the result of untwisting without the compensating writhe provided by the Phe stirrups. The structural proposal makes the clear predictions that TBP lacking the Phe stirrups will induce negative supercoiling under all conditions, and that the mutant may require negative supercoiling in order to bind at all. To test this prediction, we have made the F99A-F116A and F99A-F116A-F190AF207A site-directed TBP mutants that lack the N and C-terminal stirrups. We have characterized the binding of wild type and mutant TBP to linear DNA and to negatively supercoiled minicircles using quantitative hydroxyl radical footprinting, which is the first application for circular DNA, and electrophoretic mobility shift assays (EMSA). The results of the quantitative hydroxyl radical footprinting and EMSA suggest that mutant TBP binds better to the negatively supercoiled minicircle than to the linear DNA. We also observed quite different footprinting patterns for circular versus linear DNA at the TATA box. This indicates that the structure of TBP bound to minicircles may be different than the linear DNA. The equilibrium dissociation constants (Kd) of wild type TBP derived from hydroxyl radical footprinting titrations for the linear DNA and the -1 topoisomers of 203 bp are 11 nM and 3 nM respectively. This suggests that pre-bending of the TATA box enhanced DNA binding. From these observations, we propose that TBP binding to promoters upon gene activation may be enhanced by the topological strain induced in the DNA upon chromatin remodeling.Item COMPARATIVE PROTEOMICS STUDIES OF SOLUBLE NUCLEAR PROTEINS OF DRUG SUSCEPTIBLE AND RESISTANT HUMAN BREAST CANCER MCF-7 CELLS(2004-10-05) Fu, Zongming; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Drug resistance is a major obstacle in cancer chemotherapy. Better understanding of the mechanisms of the drug resistance can help to improve clinical treatment and develop new drugs. Since most anticancer drugs target the nuclei of the cancer cells, differential expression of nuclear proteins may play crucial roles as cancer cells acquire drug resistance. Thus I have carried out a comparative proteomics research project to study differential expression of nuclear proteins in drug resistant human breast cancer MCF-7 cells. Two dimensional gel electrophoresis and stable isotope labeling by amino acids in cell culture are used to conduct the study. Protein identification is acquired by peptide fingerprinting or microsequencing. Relative quantitation of the proteins is derived from gel comparisons and from ratios of labeled and unlabeled peptide pairs. A drug susceptible MCF-7 cell line and four drug resistant MCF-7 cell lines were examined. The drug resistant cell lines are resistant to different chemotherapeutic drugs and are well characterized. The known mechanisms of drug resistance can not satisfactorily answer how the drug resistance is conferred. One hundred and twenty proteins have been identified from the nuclear protein mixture of MCF-7 cells, from which more than 90% are classically categorized as nuclear proteins. Fourteen proteins are found to be significantly less or more abundant (more than 2 fold) in MCF-7 breast cancer cell lines resistant to etoposide, mitoxantrone, adriamycin in the presence of verapamil by both gel comparisons and isotope labeling. Abundances of cytoskeleton proteins, such as cytokeratin 8, cytokeratin 19, septin 2, and alpha tropomyosin, are altered in common across the three resistant cell lines. MCF-7 cell lines resistant to etoposide and mitoxantrone are more similar in protein abundance changes. Some of the proteins whose abundances are altered have also been reported to play important roles in resisting genotoxic stress in other normal and cancer cells. Their potential mechanistic contributions to drug resistance and implications for genetic regulation are discussed.