A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA

摘要

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 (DNAHM) substrates. Catalytic efficiency is significantly enhanced with a DNAHM substrate. Biphasic kinetic behavior during methyl incorporation is observed when unmethylated or DNAHM substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the release of enzyme from methylated DNA product. The enzyme is thermally inactivated at 30 degrees C within 20 min; this process is substantially decreased in the presence of saturating concentrations of DNAHM, suggesting that the enzyme preferentially binds DNA before S-adenosylmethionine. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order 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.