J. Flynn et al., MURINE DNA CYTOSINE-C-5 METHYLTRANSFERASE - PRE-STEADY-STATE AND STEADY-STATE KINETIC-ANALYSIS WITH REGULATORY DNA-SEQUENCES, Biochemistry, 35(23), 1996, pp. 7308-7315
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
.