title: Single-molecule analysis reveals the kinetics and physiological relevance of MutL-ssDNA binding.
abstract: DNA binding by MutL homologs (MLH/PMS) during mismatch repair (MMR) has been considered based on biochemical and genetic studies. Bulk studies with MutL and its yeast homologs Mlh1-Pms1 have suggested an integral role for a single-stranded DNA (ssDNA) binding activity during MMR. We have developed single-molecule Förster resonance energy transfer (smFRET) and a single-molecule DNA flow-extension assays to examine MutL interaction with ssDNA in real time. The smFRET assay allowed us to observe MutL-ssDNA association and dissociation. We determined that MutL-ssDNA binding required ATP and was the greatest at ionic strength below 25 mM (K(D) = 29 nM) while it dramatically decreases above 100 mM (K(D)>2 µM). Single-molecule DNA flow-extension analysis suggests that multiple MutL proteins may bind ssDNA at low ionic strength but this activity does not enhance stability at elevated ionic strengths. These studies are consistent with the conclusion that a stable MutL-ssDNA interaction is unlikely to occur at physiological salt eliminating a number of MMR models. However, the activity may infer some related dynamic DNA transaction process during MMR.
authors.affiliation: Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk, Republic of Korea.
authors.forename: Jonghyun
authors.initials: J
authors.lastname: Park
authors.forename: Yongmoon
authors.initials: Y
authors.lastname: Jeon
authors.forename: Daekil
authors.initials: D
authors.lastname: In
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
authors.forename: Changill
authors.initials: C
authors.lastname: Ban
authors.forename: Jong-Bong
authors.initials: JB
authors.lastname: Lee
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D000070956:MutL Proteins; D009154:Mutation; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins