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Originally published In Press as doi:10.1074/jbc.M200357200 on May 10, 2002

J. Biol. Chem., Vol. 277, Issue 29, 26136-26142, July 19, 2002
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Mismatch Repair in Human Nuclear Extracts
QUANTITATIVE ANALYSES OF EXCISION OF NICKED CIRCULAR MISMATCHED DNA SUBSTRATES, CONSTRUCTED BY A NEW TECHNIQUE EMPLOYING SYNTHETIC OLIGONUCLEOTIDES*

Huixian Wang and John B. HaysDagger

From the Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331

Mammalian mismatch repair (MMR) systems respond to broad ranges of DNA mismatches and lesions. Kinetic analyses of MMR processing in vitro have focused on base mismatches in a few sequence contexts, because of a lack of general and quantitative MMR assays and because of the difficulty of constructing a multiplicity of MMR substrates, particularly those with DNA lesions. We describe here simple and efficient construction of 11 different MMR substrates, by ligating synthetic oligomers into gapped plasmids generated using sequence-specific N.BstNBI nicking endonuclease, then using sequence-specific nicking endonuclease N.AlwI to introduce single nicks for initiation of 3' to 5' or 5' to 3' excision. To quantitatively assay MMR excision gaps in base-mispaired substrates, generated in human nuclear extracts lacking exogenous dNTPs, we used position- and strand-specific oligomer probes. Mispair-provoked excision along the shorter path from the pre-existing nick toward the mismatch, either 3' to 5' or 5' to 3', predominated over longer path excision by roughly 10:1 to 20:1. MMR excision was complete within 7 min, was highly specific (90:1) for the nicked strand, and was strongly mispair-dependent (at least 40:1). Nonspecific (mismatch-independent) 5' to 3' excision was considerably greater than nonspecific 3' to 5' excision, especially at pre-existing gaps, but was not processive. These techniques can be used to construct and analyze MMR substrates with DNA mismatches or lesions in any sequence context.

* This work was supported by National Institutes of Health Grant ES09848 (to J. B. H.). This is contribution 11907 from the Oregon Agricultural Experimental Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Environmental and Molecular Toxicology, Oregon State University, ALS 1007, Corvallis, OR 97331. Tel.: 541-737-1777; Fax: 541-737-0497; E-mail: haysj@bcc.orst.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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