MURINE DNA CYTOSINE-C-5 METHYLTRANSFERASE - PRE-STEADY-STATE AND STEADY-STATE KINETIC-ANALYSIS WITH REGULATORY DNA-SEQUENCES

We present the first description of K-m(DNA), K-d(DNA), k(cat), and k( methylation) for a mammalian DNA methyltransferase. Homogeneous, 190 0 00 M(r) DNA (cytosine-5-)-methyltransferase isolated from mouse erythr oleukemia cells has turnover constants of 0.15-0.59 h(-1) with single- stranded and unmethylated double-stranded oligonucleotides containing a single CpG dinucleotide. These substrates were designed to mimic DNA transcriptional cis elements previously reported to have cytosine C-5 -methylated regulation. The rate-limiting step for these substrates is the methylation step itself. In contrast, hemimethylated double-stran ded substrates show burst kinetics, consistent with a rapid methylatio n event (3 h(-1)) followed by a slower step which determines steady-st ate k(cat). Hemimethylated and unmethylated double-stranded DNA shows similar binding affinities; these results reveal the molecular basis f or the enzyme's preference for hemimethylated DNA to be the methyl tra nsfer step. Substrates with multiple recognition sites do not show bur st kinetics and have turnover rate constants of 6 h(-1). Catalytic tur nover for the mammalian enzyme is thus approximately 10-fold slower th an that for the related bacterial enzymes. Our combined results show q uantitatively that one enzyme is certainly capable of both maintenance and de novo methylation and that maintenance of the genomic methylati on pattern is preferred over the de novo establishment of new patterns . Direct comparison of the mammalian enzyme with the bacterial DNA cyt osine-C-5 methyltransferase, M.SssI, indicates dramatic differences in preferences for single-stranded, double-stranded, and hemimethylated double-stranded substrates. Moreover, the specificity hierarchy shown for the M.SssI is derived from very different changes in K-m and catal ysis than those observed for the mammalian DCMTase. These results demo nstrate that the M.SssI, and perhaps other DNA cytosine methyltransfer ases from bacteria, is functionally dissimilar to the mammalian enzyme .