MutS-DNA interactions and DNase protection analysis with surface plasmon resonance.

Abstract

Elucidation of the mechanisms of recognition of mispaired DNA by MutS and subsequent repair by the multicomponent mismatch repair machinery presents a fascinating challenge (). ATP binding or hydrolysis are implicated in several steps of the process, including enabling MutS to dissociate from the mismatch, probably through a conformational change () and for MutSL-DNA interactions (,). In addition to mismatch recognition, the protein has significant affinity for normal DNA. This may be important for the protein to scan DNA in search of a mismatch and for its proposed translocation activity. It can be expected, and especially once a MutS-DNA complex structure is solved, that numerous mutants will be generated to study the recognition mechanism. To examine stable and transient DNA interactions of the native and mutant proteins in various conformational states, assays that enable detailed kinetics are desirable. Instrumentation providing such capability is a worthwhile complement to classic assays, such as filter binding and electrophoretic mobility shift assays. Here, we describe techniques for studying the interaction of MutS and MutL with immobilized DNA using surface plasmon resonance and the BIAcore instrument. Such techniques should be readily adaptable to studies of other mismatch binding proteins (MMBP) or other DNA damage recognition proteins.