Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.
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.
Larson
Erik D
ED
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
Nickens
David
D
Drummond
James T
JT
D020137:Base Pair Mismatch / Q000235:genetics*; D020029:Base Pairing; D001483:Base Sequence; D002474:Cell-Free System; D004249:DNA Damage / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D051718:MutS Homolog 2 Protein; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured
D004270:DNA, Circular; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D009692:Nucleic Acid Heteroduplexes; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein; D011088:DNA Ligases