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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2004 - 20062

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    EXPRESSING DEINOCOCCUS RADIODURANS RECD IN ESCHERICHIA COLI: PHENOTYPIC EFFECTS IN RECBCD(-) AND RECD(-) CELLS
    (2006-12-20) polansky, steven carl; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The RecD helicase is a member of the RecBCD complex, which is essential for repair of double-stranded breaks (DSBs) in DNA via homologous recombination in Escherichia coli. The microbe Deinococcus radiodurans is capable of fixing high amounts of DSBs, owing to an efficient repair system. However, Deinococcus does not contain any recB or recC genes; only a RecD-like helicase has been observed. In Escherichia coli strains mutant for the native RecD subunit, the D. radiodurans RecD cannot restore nuclease activity when infected with T4 gene2- bacteriophage. Cell viability tests and mitomycin C exposure of RecBC(+)D(-) strains show no adverse effects from the Dr RecD. A negative phenotype was encountered with strains lacking RecBCD and expressing D. radiodurans RecD protein. Microscopy studies of RecBCD(-) E. coli expressing the D. radiodurans RecD helicase show long cellular structures termed filaments. The Dr RecD protein may be binding to and disrupting the replication fork.
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    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.