ИСТИНА

DNA mismatch repair (MMR) system maintains genome stability by correcting mismatches and small loops arising in cellular DNA. MutS is the key protein of the MMR. At the first stage of the MMR process, a protein MutS binds a mismatch and bends the DNA.The cross-linking approach combined with chromatographic techniques is useful to study conformational rearrangements of DNA-MutS complex. In this work, a cysteine residue of MutS was cross-linked to DNA fragment containing а 2'-disulfide group or 3'-terminal thiol group. The separation of reaction mixture after conjugate formation should provide the following: i) the stability of MutS homodimeric status; ii) saving the DNA-binding and ATPase activity of MutS; iii) keeping of double helix DNA structure. The protocols of exclusion HPLC on Superdex 200 (to remove the excess of DNA ) and low-pressure affinity chromatography on HiTrap Heparin column (to separate non-modified MutS) were developed for DNA-protein conjugate isolation. Purified MutS conjugated with DNA containing FRET-pair or radioactive label is able to perform ADP exchange, unbend the DNA in the ATP presence and initiate а G/T-mismatch repair.