Analysis of the human MutLalpha.MutSalpha complex.
Human DNA mismatch repair is initiated by MutSalpha which ATP-dependently recruits MutLalpha. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLalpha.MutSalpha complexes using a DNA pulldown assay. Non-specific DNA end-binding, which frequently interfered with analysis of the interaction, did not occur under the applied conditions. MutSalpha had significantly higher affinity to DNA mispairs, but its interaction with MutLalpha did not require a mismatch. Complex formation was best supported by low magnesium concentration and low temperature at physiological pH and salt concentration. Complex formation was delayed by the slowly hydrolyzable ATP analog ATPgammaS, undetectable with the non-hydrolyzable analog AMP-PNP, and occurred weakly with a combination of AMP-PNP and ADP, confirming that hydrolysis was required. The described conditions likely capture an intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites of MutSalpha.
Plotz
Guido
G
University of the Saarland, Klinik für Innere Medizin II, Homburg/Saar, Germany. guido.plotz@uniklinik-saarland.de
Piiper
Albrecht
A
Wormek
Marc
M
Zeuzem
Stefan
S
Raedle
Jochen
J
D000255:Adenosine Triphosphate / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D020137:Base Pair Mismatch; D001665:Binding Sites; D019921:Biotinylation; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D008954:Models, Biological; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D012492:Salts / Q000737:chemistry / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D017931:DNA Primers; C500929:MutLalpha protein, human; D009363:Neoplasm Proteins; D009711:Nucleotides; D012492:Salts; D000266:Adenylyl Imidodiphosphate; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D045643:DNA Repair Enzymes; D008274:Magnesium