TAILIEUCHUNG - Báo cáo khoa học: 2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase–substrate interaction

EcoRII DNA methyltransferase () recognizes the 5¢ CC*T/AGG 3¢ DNA sequence and catalyzes the transfer of the methyl group fromS-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). Here, we study themechanismof byDNA containing 2-pyrimidinone, a cytosine analogue lacking an NH2 group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts with functional groups of pyrimidine bases of the recognition sequence | Eur. J. Biochem. 271 2391-2399 2004 FEBS 2004 doi 2-Pyrimidinone as a probe for studying the froRII DNA methyltransferase-substrate interaction Oksana M. Subach1 Anton V. Khoroshaev1 Dmitrii N. Gerasimov1 Vladimir B. Baskunov1 Anna K. Shchyolkina2 and Elizaveta S. Gromova1 1 Chemistry Department Moscow State University Russia 2Engelghardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia EcoRII DNA methyltransferase recognizes the 5 . .CC T AGG. .3 DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue C . Here we study the mechanism of inhibition of by DNA containing 2-pyrimidinone a cytosine analogue lacking an NH2 group at the C4 position of the pyrimidine ring. Also DNA containing 2-pyrimidinone was used for probing contacts of with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5 .CCT sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and . 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with . In the absence of the cofactor S-adenosyl-L-methionine proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of .

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