Аннотация:Four-stranded non-canonical forms of DNA – G-quadruplexes (G4) – perform regulatory functions supporting vital cell processes. However, they contribute to genome instability alongside. One of the molecular mechanisms countering DNA lesions is mismatch repair system (MMR) which corrects the mistakes of DNA polymerase and therefore serves to genomic integrity. E. coli MMR system includes three key proteins: MutS which is responsible for misincorporated nucleotides recognition; MutL that recruits MutH protein to introduce single-strand cut. MutS from E. coli is capable of specific and efficient tetramolecular G4 binding (Ehrat et al., 2012). Nevertheless, an additional information is required to comprehend the actual role and mechanism of interaction between MutS and G4. Moreover, there is no available data on the interaction of G4 with MutL and MutH proteins. In order to investigate the influence of G4 on the initial steps of MMR, we suggested 76 bp DNA model system which included the GT mismatch and monomethylated 5' Gm6ATC 3'/3' CTAG 5' MutH recognition site divided by the sequence containing (GGGT)4-motif forming parallel intramolecular G4. The protection of guanines belonging to the G4-motif from the modification in dimethylsulphate footprinting and the presence of detached signals in 10-12 ppm region of 1H NMR spectra allowed us to confirm the formation of G4 structure. The binding of MutS to intramolecular G4 DNA in conditions providing different MutS conformations was investigated. We demonstrated that the difference between MutS affinity to G4 and other DNA molecules greatly increased upon ATP or ATPγS addition which was consistent with the results for tetramolecular G4 DNA (Ehrat et al., 2012). It was shown for the first time that MutL protein binds to 76 bp DNA with G4. The examination of MutH cleavage efficiency of proposed DNA system alone and in the presence of protein cofactors (E. coli MutS and MutL) demonstrated no dependence on G4 in DNA. Hence, in this work for the first time the interaction between G4 and MutS-MutL-MutН complex was investigated, yet the influence of G4 DNA on MMR functioning is still not obvious.
