Nucleotide dependent conformational changes of MutL.

Abstract

MutL is one of the enzymes required for mismatch repair (MMR). Mutations in MutL are linked to Lynch Syndrome, which predisposes individuals to colorectal and other cancers. In MMR, MutL is known to nick the error-containing daughter strand of mismatch DNA as well as form complexes with other MMR proteins such as MutS. MutL accomplishes this by undergoing a series of conformational changes which include extended, one-arm, semi-condensed, and condensed states in the presence of ATP. These dynamic conformational changes are needed to coordinate MMR, but the exact sequence of events and the impact of disease-associated mutations are unknown. We used site-directed mutagenesis to fluorescently label sites in the different domains of Thermus Aquaticus MutL, including the N-terminal, linker arms, and C-terminal domains. We expressed MutL variants with wild-type and ATPase-deficient activity and monitored their dynamic conformational changes under differing nucleotide conditions with pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) in solutions of freely diffusing molecules. Using the same technique, we also monitored the interactions of MutL with MutS, the enzyme that initially recognizes a mismatch, to reveal the specific interactions and conformational changes that are necessary to initiate mismatch repair.