ИСТИНА

DNA duplexes containing a disulfide group are widely used in biochemical studies. A disulfide group in DNA selectively reacts with a cysteine residue of a protein under physiological conditions with a high yield that is why such modified duplexes can be employed to map protein contacts with DNA, as well as to analyse the structure of DNA–protein complexes. Many metabolic processes are based on formation of large macromolecular complexes, which change their conformation while functioning. One of them is DNA mismatch repair (MMR). Three proteins, MutS, MutL and MutH, are important to perform mismatch recognition and strand discrimination, which is required for accurate initiation of DNA mismatch repair in E. coli. MMR is initiated after a mismatch is recognised and bound by MutS. Mismatch binding induces conformational changes in MutS, resulting in recruitment of MutL. MutH introduces a break into the “erroneous” and transiently unmethylated daughter strand and initiates the subsequent stages of the repair process. We have suggested to use DNA with a disulfide group to covalently bind a certain conformation of the MutS and MutL complex. Based on the crystal structure of MutS with DNA, we have engineered MutS mutants, each containing a single cysteine residue in a position that would be close to DNA upon binding. The modified DNAs for cross-linking to a single-cysteine MutS contain disulfide groups introduced at the heterocyclic base at the distance of 7 bp away from the mismatch. We used the linkers of different lengths between the reactive group and the DNA to test whether MutS in the cross-linked complex is capable of conformational transitions. We obtained and purified MutS-DNA covalent conjugates with high yield, and used them to investigate the ability of MutS to bend and unbend DNA, as the evidence of its activity. To facilitate the detection of such changes in DNA conformation, we have introduced, in addition to the disulfide group, two fluorophores forming a FRET pair, placing them in one DNA strand, but on both sides of the mismatch. We have observed that MutS is active when conjugated with DNA. For the first time, the rate of DNA unbending in the cross-linked complex has been shown to depend on ATP concentration. We have also measured the kinetics of conformational changes in DNA using a stopped-flow technique. Moreover, the ability of MutS (when conjugated with DNA) to recruit MutL has been tested. We have found that MutL is able to form a stable complex with MutS-DNA conjugates. Kinetics of MutL recruitment to the cross-linked complex has been measured. We have shown that MutL in a “closed” conformation forms a complex with MutS faster and more efficiently than in an “open” conformation. To detect the contact sites between MutL and DNA, the DNAs with disulfide groups have been tested. MutL-DNA conjugates have been obtained with high yield. Cys218 and Cys251 of MutL are the closest to the DNA ligand; MutS presence increases the yield of the covalent conjugate. Thus, covalent binding of MutS and MutL to DNA can be used as a strategy for structural studies of MutS/L/H complex with DNA, to investigate the mechanism of mismatch repair initiation.