Analysis of the human MutLα·MutSα complex

Abstract Human DNA mismatch repair is initiated by MutSα which ATP-dependently recruits MutLα. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLα·MutSα 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. MutSα had significantly higher affinity to DNA mispairs, but its interaction with MutLα 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 ATPγS, 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 MutSα.