ИСТИНА

Mismatch repair (MMR) maintains genome stability by correcting mismatches and small insertion/deletion loops introduced by DNA polymerases. At the first stage of the MMR process, a protein named MutS binds a mismatch and bends the DNA. ATP binding by MutS allows its conformational change into a sliding clamp form which is required for the downstream MMR stages. In this work, MutS crosslinking to DNA was used to fix transient protein conformations during the MMR process. A protocol of the crosslinking followed by complex purification was developed. The DNA fragment contained a disulfide group on a linker capable of reacting with a cysteine residue of the protein. Different lengths of the linkers were used in order to test whether and how constraints by the crosslink to DNA influence the conformational transition of MutS into the sliding clamp state. The DNA fragment contained a donor and acceptor fluorophore to allow monitoring the DNA bending and unbending in the crosslinked complex using FRET. Kinetics of the DNA conformational changes were measured by stopped-flow technique. The rate of the DNA unbending in the crosslinked complex was shown to depend on the ATP concentration.