Аннотация:The DNA mismatch repair (MMR) pathway removes errors that escaped from polymerase proofreading activity during replication. MMR corrects DNA by excising an extended single-stranded fragment of the newly synthesized DNA and then filling the resulting gap. MutS and MutL are the key MMR proteins responsible for mismatch recognition and initiation of the daughter strand nicking. To date, there is no uniform opinion about the structure of the protein-protein and DNA-protein complexes and the mechanism of their action during repair initiation.
In this work, we have suggested a method of crosslinking MutS and MutL from E.coli with DNA for investigation of MMR mechanism. We designed modified DNA that contain a disulfide group for protein crosslinking and in number of cases fluorophores (for FRET studies). We obtained and purified covalent complex MutS-DNA and have shown that MutS remains active in the conjugate. The rate of DNA unbending is strongly ATP-dependent. Moreover, MutS-DNA covalent complex is able to recruit MutL. We investigated the kinetics of complex formation between MutL and different MutS-DNA conjugates. MutS-DNA conjugate form complex with MutL much more efficiently when the duplex is fixed in the DNA-binding region of MutS.
For the first time, covalently bound complexes of MutL E.coli with DNA have been obtained with high yield. We have shown that amino acid residues 218 and 251 of this MutL are the closest ones to the DNA ligand. Presence of MutS increases the yield and the rate of conjugate formation between MutL variants and modified DNA. For the first time we applied crosslinking approach for MutL protein with endonuclease function from N. gonorrhoeae and demonstrated the formation of DNA-protein conjugate. It was suggested that Cys residues of endonuclease motif form the bound with DNA. We showed that the yield of crosslinking depends on zinc ions presence. The covalent fixation of MMR proteins on DNA could help for better understanding of MutS-MutL-DNA complex structure that makes different conformation during MMR process.
