A CELL-CYCLE-REGULATED ADENINE DNA METHYLTRANSFERASE FROM CAULOBACTER-CRESCENTUS PROCESSIVELY METHYLATES GANTC SITES ON HEMIMETHYLATED DNA

The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated DNA (DNA(HM)) substrat es. Catalytic efficiency is significantly enhanced with a DNA(HM) subs trate. Biphasic kinetic behavior during methyl incorporation is observ ed when unmethylated or DNA(HM) substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the re lease of enzyme from methylated DNA product. The enzyme is thermally i nactivated at 30 degrees C within 20 min; this process is substantiall y decreased in the presence of saturating concentrations of DNA(HM), s uggesting that the enzyme preferentially binds DNA before S-adenosylme thionine. The activity of the enzyme shows an unusual sensitivity to s alt levels, apparently dissociating more rapidly from methylated DNA p roduct as the salt level is decreased. The enzyme acts processively du ring methylation of specific DNA sequences, indicating a preferred ord er of product release in which S-adenosylhomocysteine is released from enzyme before fully methylated DNA. The kinetic behavior and activity of the enzyme are consistent with the temporal constraints during the cell cycle-regulated methylation of newly replicated chromosomal DNA.