Human DNA mismatch repair is initiated by MutS alpha which ATP-dependently recruits MutL alpha. Analysis of this complex is difficult due to its transient and dynamic nature, We have optimized conditions for investigation OF MutL alpha:MutS alpha 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. MutS alpha had significantly higher affinity to DNA mispairs, but its interaction with MutL alpha. 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 ATP gamma S, 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 ail intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites Of MutS alpha. (c) 2005 Elsevier Inc. All rights reserved.