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The mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the potential repair of the most abundant oxidised pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) by E. coli MMR system has been investigated. In a partially reconstituted MMR system using MutS/MutL/MutH proteins we observed that G/Tg-containing plasmid DNA was impaired to provoke incision and thereby initiation of MMR. Compared to this DNA with A/Tg pair could be a better but still poor substrate for MMR system. To reveal which steps in the initial phase of the MMR were affected, the interactions of MutS with A/Tg- and G/Tg-containing DNAs were investigated in details. MutS had a 3-6 times lower affinity to Tg-containing DNAs compared to G/T-duplex (the Kd was ~7.2 nM). The Kd value for duplex containing G/Tg pair (~45 nM) and A/Tg-duplex (~25 nM) were similar as for the canonical DNA (~33 nM). The higher affinity of MutS to A/Tg-duplex is in agreement with the slightly higher DNA incision by MutH/MutL/MutS complex as compared with native plasmid. Using a recently developed FRET assay to monitor DNA kinking and DNA binding orientation by MutS, we show that despite of reduced affinity to oxidised DNA lesion the MutS orientation on the DNAs with G/Tg or A/Tg pair and with G/T mismatch was the same. However, in the case of both Tg-containing DNAs the characteristic DNA kink was not observed. The lack of such a kinking can be explained by the disruption of specific DNA binding with MutS. Furthermore DNAs containing G/Tg and A/Tg pairs did not stimulate the nucleotide exchange in ATPase domain of MutS as effective as that observed for DNA with a G/T pair. In summary, the MutS transformation to an active conformation (sliding clamp) and hence the interaction with MutL is likely impaired. Taken together, our results demonstrate that thymidine glycol residue in DNA probably not repaired/processed (or repaired only with a small extent) by the MMR system. The lack of repair can be attributed to the failure of MutS to interact with modified DNA containing nonplanar thymine glycol.