Nucleic Acids Research Advance Access originally published online on October 4, 2007
Nucleic Acids Research 2007 35(20):6727-6739; doi:10.1093/nar/gkm734
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Nucleic Acids Research, 2007, Vol. 35, No. 20 6727-6739
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Human DNA mismatch repair: coupling of mismatch recognition to strand-specific excision
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis OR 97331-7301, USA
*To whom correspondence should be addressed. Tel: +1 541 737 1777; Fax: +1 541 737 0497; Email: haysj{at}science.oregonstate.edu
Received April 16, 2007. Revised August 23, 2007. Accepted September 4, 2007.
Eukaryotic mismatch-repair (MMR) proteins MutS
and MutL couple recognition of base mismatches to strand-specific excision, initiated in vivo at growing 3' ends and 5' Okazaki-fragment ends or, in human nuclear extracts, at nicks in exogenous circular substrates. We addressed five biochemical questions relevant to coupling models. Excision remained fully efficient at DNA:MutS ratios of nearly 1 to 1 at various mismatch-nick distances, suggesting a requirement for only one MutS molecule per substrate. As the mismatch-nick DNA contour distance D in exogenous substrates increased from 0.26 to 0.98 kbp, initiation of excision in extracts decreased as D–0.43 rather than the D–1 to D–2 predicted by some translocation or diffusion models. Virtually all excision was along the shorter (3'–5') nick-mismatch, even when the other (5'–3') path was less than twice as long. These observations argue against stochastically directed translocating/diffusing recognition complexes. The failure of mismatched DNA in trans to provoke excision of separate nicked homoduplexes argues against one-stage (concerted) triggering of excision initiation by recognition complexes acting through space. However, proteins associated with gapped DNA did appear to compete in trans with those in cis to mismatch-associated proteins. Thus, as in Escherichia coli, eukaryotic MMR may involve distinct initial-activation and excision-path-commitment stages.