Nucleic Acids Research, 2002, Vol. 30, No. 3 e14
© 2002 Oxford University Press
Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro
Department of Biology, Indiana University, Bloomington, IN 47405, USA
The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G·T mismatch was achieved by placing a nick 10–40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.
* To whom correspondence should be addressed. Tel: +1 812 856 4184; Fax: +1 812 855 6705; Email: jdrummon{at}bio.indiana.edu