Detection of high-affinity and sliding clamp modes for MSH2-MSH6 by single-molecule unzipping force analysis.
Mismatch repair (MMR) is initiated by MutS family proteins (MSH) that recognize DNA mismatches and recruit downstream repair factors. We used a single-molecule DNA-unzipping assay to probe interactions between S. cerevisiae MSH2-MSH6 and a variety of DNA mismatch substrates. This work revealed a high-specificity binding state of MSH proteins for mismatch DNA that was not observed in bulk assays and allowed us to measure the affinity of MSH2-MSH6 for mismatch DNA as well as its footprint on DNA surrounding the mismatch site. Unzipping analysis with mismatch substrates containing an end blocked by lac repressor allowed us to identify MSH proteins present on DNA between the mismatch and the block, presumably in an ATP-dependent sliding clamp mode. These studies provide a high-resolution approach to study MSH interactions with DNA mismatches and supply evidence to support and refute different models proposed for initiation steps in MMR.
Jiang
Jingjing
J
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Bai
Lu
L
Surtees
Jennifer A
JA
Gemici
Zekeriyya
Z
Wang
Michelle D
MD
Alani
Eric
E
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D020137:Base Pair Mismatch; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008863:Microspheres; D051718:MutS Homolog 2 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000032:analysis; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013314:Stress, Mechanical
D004268:DNA-Binding Proteins; C098790:MSH6 protein, S cerevisiae; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C489751:MSH2 protein, S cerevisiae; D051718:MutS Homolog 2 Protein