ИСТИНА

Mismatch repair (MMR) maintains genome stability by correcting mismatches and small insertion/deletion loops. MutS binds a mismatch and bends the DNA at the first stage of the MMR process. ATP-binding results in a series of ill-defined large conformational changes leading to a stable sliding clamp state of MutS which required for the downstream MMR stages. Guided by the co-crystal structures of MutS bound to DNA with mismatches we designed a site-specific crosslinking and purification procedures for the complex between DNA and MutS. The modified DNAs for crosslinking to a single-cysteine residue in MutS contain asymmetrical disulfide groups introduced at the phosphate or the heterocyclic base. The linkers of different lengths between reactive group and DNA were used to test whether MutS in the cross-linked complex is capable of conformational transitions. The influence of MutS binding on bending/unbending of donor and acceptor fluorophore labeled DNA was monitoring using FRET assay. Moreover, the ability of MutS to recruit MutL was tested. Kinetics of the DNA conformational changes were measured by stopped-flow technique. The rate of the DNA unbending in the cross-linked complex was shown to depend on the ATP concentration.