2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.
EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 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 M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' 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 M.EcoRII 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 DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. 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 S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.
Subach
Oksana M
OM
Chemistry Department, Moscow State University, Russia.
Khoroshaev
Anton V
AV
Gerasimov
Dmitrii N
DN
Baskunov
Vladimir B
VB
Shchyolkina
Anna K
AK
Gromova
Elizaveta S
ES
D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015394:Molecular Structure; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D011744:Pyrimidinones / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D013941:Thymine / Q000737:chemistry
D011744:Pyrimidinones; D003596:Cytosine; D004247:DNA; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D013941:Thymine