Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair
- †Department of Biochemistry and
- ‡Howard Hughes Medical Institute, Box 3711, Duke University Medical Center, Durham, NC 27710
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Contributed by Paul Modrich, June 1, 2007 (received for review May 21, 2007)
Abstract
The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3′-to-5′ or 5′-to-3′ excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRIE111Q, a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70–80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRIE111Q away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour.
Footnotes
- §To whom correspondence should be addressed. E-mail: modrich{at}biochem.duke.edu
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Author contributions: A.P. and P.M. designed research; A.P. performed research; A.P. analyzed data; and A.P. and P.M. wrote the paper.
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Conflict of interest statement: P.M. serves on the Scientific Advisory Board of Codon Devices; however, this paper is completely unrelated to his role in the company.
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Freely available online through the PNAS open access option.
- © 2007 by The National Academy of Sciences of the USA
