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Title	Structure-function studies of late stages of E. Coli MMR : interaction of DNA helicase II with single-stranded DNA binding protein (SSB) and MutL
Creator	In, Junghoon.
Abstract	DNA mismatch repair (MMR) is the post-replicative mechanism by which errors made during DNA replication and recombination are corrected. E. coli UvrD (DNA helicase II) is involved in mismatch repair, where it unwinds dsDNA in order to correct DNA errors following DNA incision by the combined activation of MutS-MutL-MutH. Using Atomic Force Microscopy, I was able to demonstrate that UvrD interacts with SSB, which stimulates UvrD-catalyzed unwinding of double-nicked circular DNA. This result suggests that the interaction of SSB with UvrD facilitates unwinding of DNA substrates. Deletion of acidic 10 residues of the C-terminus results in a mutant, SSBdeltaC10, that neither interacts with UvrD nor stimulates the function of UvrD, indicating that the SSB C-terminus mediates SSB interaction with UvrD. WtSSB recruits UvrD onto a nick of the DNA substrate to initiate unwinding of DNA, as demonstrated by wtSSB reducing UvrD binding to blunt-ended and 3' overhang DNA. I observed that wtSSB facilitates UvrD-catalyzed unwinding of nicked DNA in a 3-5' direction. These findings suggest that the stimulatory effect of SSB on UvrD helicase activity is not solely due to SSB's binding function to ssDNA, but due to the specific interaction of SSB with UvrD. I have shown that UvrD also interacts with MutL. The interaction of UvrD and MutL facilitates the loading of UvrD in the presence of ATP onto duplex DNA including 3-nt 3' overhang, suggesting that MutL increases the movement of UvrD along DNA. Additionally, ATP and a nonhydrolyzable ATP analog (AMPPNP) reduced UvrD binding to end of 3' overhang DNA. Taken together, these findings suggest that UvrD mediates the late stage of MMR machinery by the interaction with SSB and MutL. It is these interactions that facilitate the processivity of UvrD unwinding of DNA during repair to maintain genomic stability.
Repository	University of North Carolina at Chapel Hill. Library
Faculty Advisor	Erie, Dorothy A., 1959-
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	Applied and Materials Sciences
Department	Applied and Materials Sciences
Digital Collection	UNC Electronic Theses and Dissertations
Host	University of North Carolina at Chapel Hill
File Size	4.24 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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Structure-function studies of late stages of E. Coli MMR : interaction of DNA helicase II with single-stranded DNA binding protein (SSB) and MutL

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