The PvuII DNA (cytosine-N4)-methyltransferase comprises two trypsin-defined domains, each of which binds a molecule of S-adenosyl-L-methionine.
Earlier studies have shown that PvuII methyltransferase is monomeric and transfers a methyl group from S-adenosyl-l-methionine (AdoMet) to cytosine, generating N4-methylcytosine in duplex 5'-CAGCTG-3' DNA. This study examines the interactions between PvuII methyltransferase and AdoMet. Trypsin preferentially cleaved the protein into two large fragments, with initial cleavages after Arg183 and Lys186. UV-mediated photochemical labeling with [3H-CH3]AdoMet, followed by trypsin digestion, revealed that both large fragments of the protein were labeled. Rapid gel filtration confirmed that each molecule of the intact enzyme bound two molecules of AdoMet (net Kd = 9.3 microM). When PvuII methyltransferase was preincubated with a range of [3H-CH3]AdoMet concentrations, bursts of product formation resulted upon DNA addition. These data indicate that PvuII methyltransferase is catalytically competent with one and with two bound molecules of AdoMet. These results, together with those from earlier studies, suggest possible roles for the second molecule of AdoMet.
Adams
G M
GM
Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43699-0008, USA.
Blumenthal
R M
RM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D014466:Ultraviolet Rays
D010446:Peptide Fragments; D012436:S-Adenosylmethionine; D004247:DNA; C047084:DNA modification methylase PvuII; D015257:DNA-Cytosine Methylases; D014357:Trypsin