Murine DNA cytosine-C-5 methyltransferase: Pre-steady- and steady-state kinetic analysis with regulatory DNA sequences
Murine DNA cytosine-C-5 methyltransferase: Pre-steady- and steady-state kinetic analysis with regulatory DNA sequences
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DOI：
10.1021/bi9600512
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发表时间：
1996-06-11
期刊：
影响因子：
2.9
通讯作者：
Reich, NO
中科院分区：
文献类型：
--
作者：
Flynn, J;Glickman, JF;Reich, NO
我们首次描述了哺乳动物 DNA 甲基转移酶的 K-m(DNA)、K-d(DNA)、k(cat) 和 k(甲基化)。从小鼠红白血病细胞中分离出的均质 190 000 M(r) DNA（胞嘧啶-5-）-甲基转移酶，其转换常数为 0.15-0.59 h(-1)，与含有单个 CpG 二核苷酸的单链和未甲基化双链寡核苷酸。这些底物被设计来模拟先前报道的具有胞嘧啶 C-5 甲基化调节的 DNA 转录顺式元件。这些底物的限速步骤是甲基化步骤本身。相反，半甲基化双链底物表现出爆发动力学，与快速甲基化事件（3 h(-1)）一致，随后是确定稳态 k(cat) 的较慢步骤。半甲基化和非甲基化双链 DNA 表现出相似的结合亲和力；这些结果揭示了该酶偏爱半甲基化 DNA 作为甲基转移步骤的分子基础。具有多个识别位点的底物不显示突发动力学并且具有6 h(-1) 的周转率常数。因此，哺乳动物酶的催化周转比相关细菌酶的催化周转慢大约 10 倍。我们的综合结果定量地表明，一种酶肯定能够维持和从头甲基化，并且基因组甲基化模式的维持优于从头建立新模式。哺乳动物酶与细菌 DNA 胞嘧啶-C-5 甲基转移酶 M.SssI 的直接比较表明，对单链、双链和半甲基化双链底物的偏好存在显着差异。此外，M.SssI 显示的特异性层次源自与哺乳动物 DCMTase 观察到的非常不同的 K-m 和催化变化。这些结果表明，M.SssI，或许还有来自细菌的其他 DNA 胞嘧啶甲基转移酶，在功能上与哺乳动物酶不同。
We present the first description of K-m(DNA), K-d(DNA), k(cat), and k(methylation) for a mammalian DNA methyltransferase. Homogeneous, 190 000 M(r) DNA (cytosine-5-)-methyltransferase isolated from mouse erythroleukemia 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-stranded substrates show burst kinetics, consistent with a rapid methylation event (3 h(-1)) followed by a slower step which determines steady-state k(cat). Hemimethylated and unmethylated double-stranded DNA shows similar binding affinities; these results reveal the molecular basis for the enzyme's preference for hemimethylated DNA to be the methyl transfer step. Substrates with multiple recognition sites do not show burst kinetics and have turnover rate constants of 6 h(-1). Catalytic turnover for the mammalian enzyme is thus approximately 10-fold slower than that for the related bacterial enzymes. Our combined results show quantitatively that one enzyme is certainly capable of both maintenance and de novo methylation and that maintenance of the genomic methylation pattern is preferred over the de novo establishment of new patterns. Direct comparison of the mammalian enzyme with the bacterial DNA cytosine-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 catalysis than those observed for the mammalian DCMTase. These results demonstrate that the M.SssI, and perhaps other DNA cytosine methyltransferases from bacteria, is functionally dissimilar to the mammalian enzyme.