Kinetic Characterization and Domain Analysis of the helicase RecD2 from Deinococcus radiodurans

dc.contributor.advisorJulin, Douglas Aen_US
dc.contributor.authorShadrick, William Roberten_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.description.abstractThe gram positive bacterium <italic>D. radiodurans</italic> is known for its extreme resistance to radiation and an extraordinary ability to reconstitute its genome after sustaining large numbers of double strand breaks (DSB's). Genome analysis does not immediately reveal a biochemical basis for this incredible DNA repair ability. In <italic>E. coli</italic>, DSB's are mainly repaired through the RecBCD pathway via homologous recombination. The <italic>D. radiodurans</italic> genome contains no known homologues of RecB or RecC, but sequence analysis has identified a homologue of RecD, termed RecD2. The function of RecD2 in <italic>D. radiodurans</italic> is unknown, as RecD elsewhere has only been found as a component of the RecBCD complex. Our research has focused on biochemical characterization of RecD2. Previous work in our lab established that RecD2 is a DNA helicase with limited processivity and a preference for forked substrates. We have studied the unwinding mechanism of the enzyme, as measured by rates of DNA unwinding and behavior on various substrates. Reactions conducted under single turnover conditions have allowed us to determine the processivity and the step size of RecD2. RecD2 pre-bound to dsDNA substrate is capable of unwinding 12 bp, but not 20 bp, when excess ssDNA is added to prevent rebinding of enzyme to substrate. Unwinding of the 12 bp substrate under single turnover conditions could be modeled using a two step mechanism, with k<sub>unw</sub> = 5.5 s<super>-1</super> and dissociation from partially unwound substrate k<sub>off</sub> = 1.9 s<super>-1</super>. Results derived from these rate constants indicate an unwinding rate of 15-20 bp/ sec, with relatively low processivity (P = 0.74). Glutaraldehyde cross-linking showed formation of multimers of RecD2 in the absence of DNA, but this was not detectable by size exclusion chromatography. We were able to separate the N-terminal region from the helicase core of RecD2 using limited proteolysis. It was not possible to characterize the C-terminal helicase domain due to its low solubility upon overexpression in <italic>E. coli</italic>.en_US
dc.subject.pqcontrolledChemistry, Biochemistryen_US
dc.subject.pquncontrolledDNA repairen_US
dc.subject.pquncontrolledstep sizeen_US
dc.titleKinetic Characterization and Domain Analysis of the helicase RecD2 from Deinococcus radioduransen_US
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