DNA template requirements for human mismatch repair in vitro.
The human mismatch repair pathway is competent to correct DNA mismatches in a strand-specific manner. At present, only nicks are known to support strand discrimination, although the DNA end within the active site of replication is often proposed to serve this role. We therefore tested the competence of DNA ends or gaps to direct mismatch correction. Eight G.T templates were constructed which contained a nick or gap of 4, 28, or approximately 200 nucleotides situated approximately 330 bp away in either orientation. A competition was established in which the mismatch repair machinery had to compete with gap-filling replication and ligation activities for access to the strand discontinuity. Gaps of 4 or 28 nucleotides were the most effective strand discrimination signals for mismatch repair, whereas double strand breaks did not direct repair to either strand. To define the minimal spatial requirements for access to either the strand signal or mismatch site, the nicked templates were linearized close to either site and assayed. As few as 14 bp beyond the nick supported mismatch excision, although repair synthesis failed using 5'-nicked templates. Finally, asymmetric G.T templates with a remote nick and a nearby DNA end were repaired efficiently.
Iams
Keith
K
Department of Biology, Indiana University, 1001 E Third Street, Bloomington, IN 47405, USA.
Larson
Erik D
ED
Drummond
James T
JT
D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry*; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D051718:MutS Homolog 2 Protein; D011518:Proto-Oncogene Proteins / Q000502:physiology; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D011518:Proto-Oncogene Proteins; D004247:DNA; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein