Hmi1p and UvrD : two Superfamily I helicases and their respective roles in S. cerevisiae mtDNA maintenance and E. coli methyl-directed mismatch repair

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Title	Hmi1p and UvrD : two Superfamily I helicases and their respective roles in S. cerevisiae mtDNA maintenance and E. coli methyl-directed mismatch repair
Creator	Monroe, Danny Scott.
Abstract	Helicases are vital to all aspects of genomic maintenance. Double-stranded DNA, DNA/RNA hybrids, and self-annealed RNA require the action of this class of proteins to separate into single-stranded components that can be manipulated for cellular function and survival. Hmi1p, a Superfamily I helicase shown to localize to the mitochondria, is required to maintain mtDNA in Saccharomyces cerevisiae. Through biochemical studies, we are able to show that Hmi1p is a nucleic acid stimulated NTPase that unwinds DNA in a 3-5' direction. Furthermore, we show that site-directed mutagenesis of the conserved lysine residue of the Walker A box effectively obliterates ATP hydrolysis and, therefore, helicase activity. Suprisingly, genetic analyses indicate that this ATP hydrolysis mutant is able to partially complement the petite phenotype associated with deltahmi1 mutants. A possible structural role as part of the mtDNA replication machinery and future experimentation is discussed. The Escherichia coli Superfamily I helicase, UvrD, is involved in replication, nucleotide excision repair, and methyl-directed mismatch repair (MMR). The role of UvrD in MMR is well-defined but the mechanism of unwinding stimulation by the protein MutL is poorly understood. We have constructed and tested a substrate designed to track relative distances of MutL and UvrD along a dsDNA region through the use of single-molecule FRET (smFRET) and total internal reflection microscopy (TIRM). We show that UvrD and MutL bind the substrate separately and, when combined, enhance the binding of both proteins to the DNA. An in-depth discussion of necessary controls and experiments is expanded upon to unravel this mechanism.
Repository	University of North Carolina at Chapel Hill. Library
Faculty Advisor	Matson, Steve.
Date (Graduated)	2008-12
Date (Title Page)	2008
Form General	Dissertations
Type (Thesis or Dissertation)	Dissertation
Language	English
Degree Name	Doctor of Philosophy
Degree Level	Ph. D.
Discipline	Biology
Department	Biology
Digital Collection	UNC Electronic Theses and Dissertations
Host	University of North Carolina at Chapel Hill
File Size	6.2 MB
Copyright Holder	Author
Usage Rights	The author has granted the University of North Carolina at Chapel Hill a limited, non-exclusive right to make this publication available to the public. The author retains all other rights.

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Hmi1p and UvrD : two Superfamily I helicases and their respective roles in S. cerevisiae mtDNA maintenance and E. coli methyl-directed mismatch repair

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