ИСТИНА

The DNA mismatch repair (MMR) system is very conservative among of every life things kingdoms. Many details have been illuminated about the MMR. A key protein of the system, MutS, has been investigated with crystallographic studies and biochemical analyses. But it still is unknown how MutS interact with DNA and other components of the system after mismatch is recognized. The particular aim is to investigate how MutS discriminates between homo- and heteroduplexes of DNA and to display features of the complex structure between MutS and homoduplex. Cross-linking between MutS and DNA was suggested for crystallographic and biochemical studies. We propose to carry out cross-linking experiments with oligonucleotides containing 2’-iodoacetamide function and thio-containing as reactive DNAs, and single-cysteine (SC) MutS. We have analyzed the crystal structure of the MutS in complex with heteroduplex. We replaced 3 amino acids (468,469, 497) which are interact with the DNA by cysteine. All of them belong to the clamp domain of MutS. We have purified the WT MutS and mutant forms: CF, SC 468, 469, 497. The yield of cross-linking reaction between SCMutS and DNA duplexes containing single 2'-iodoacetamide function with or without mismatch was up to 10%. Reaction yield of cross-linking reached maximum possible (50%) in case SCMutS(N497C) with DNA duplex containing mismatch and thio-group. The variant is most promising for subsequent studies.