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G-quadruplexes in DNA (G4) have recently gravitated the attention of researches. Being widespread in genomes,especially in promoter regions and telomeres, they perform regulatory functions supporting vital cell processes. However, G4 DNA is supposed to contribute to genome instability alongside. Mismatch repair system (MMR) is among mechanisms summoned to cope with DNA lesions. Latter evidence of interaction between G4 and one of key MMR proteins MutS has revealed significant differences in binding mode as compared to specific MutS substrate – DNA with a mismatch. Nevertheless, 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 between G4 and other MMR proteins – MutL and MutH. In order to investigate the influence of G4 on the initial steps of MMR, we suggested DNA model system that included the motif of parallel intramolecular G4 flanked with duplex regions carrying MutH recognition site and GT-mismatch. The presence of G4 structure in conditions employed in further experiments was confirmed by dimethylsulphate footprinting and 1H NMR. Applying gel mobility shift assay we investigated the binding of MutS from E. coli to G4 DNA in conditions providing different MutS conformations. Not significant in the presence of ADP, the difference between MutS affinity to G4 and other DNA molecules greatly increased upon ATP or ATPγS addition.Thereafter the proposed G4 DNA were subjected to hydrolysis induced by E. coli MutH. The hydrolysis efficiency in the presence of protein cofactors (E. coli MutS and MutL) was demonstrated to be not dependent on G4 presence in DNA duplex. Hence, in this work for the first time the interaction between G4 and MMR as a whole was investigated, yet the influence of G4 DNA on MMR functioning is still not obvious