Murine DNA Cytosine-C5 Methyltransferase: Pre-Steady- and Steady-State Kinetic Analysis with Regulatory DNA Sequences

详细信息    查看全文

• 作者：James Flynn ; J. Fraser Glickman ; and Norbert O. Reich
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：June 11, 1996
• 年：1996
• 卷：35
• 期：23
• 页码：7308 - 7315
• 全文大小：552K
• 年卷期：v.35,no.23(June 11, 1996)
• ISSN：1520-4995

文摘

We present the first description ofK_m^DNA,K_d^DNA, k_cat,and k_methylation for a mammalianDNAmethyltransferase. Homogeneous, 190 000 M_rDNA (cytosine-5-)-methyltransferase isolated from mouseerythroleukemia cells has turnover constants of 0.15-0.59h^-1 with single-stranded and unmethylateddouble-stranded oligonucleotides containing a single CpG dinucleotide.These substrates were designedto mimic DNA transcriptional cis elements previously reported to havecytosine C-5-methylated regulation.The rate-limiting step for these substrates is the methylationstep itself. In contrast, hemimethylated double-stranded substrates show burst kinetics, consistent with a rapidmethylation event (3 h^-1) followed by aslower step which determines steady-state k_cat.Hemimethylated and unmethylated double-stranded DNAshows similar binding affinities; these results reveal the molecularbasis for the enzyme's preference forhemimethylated DNA to be the methyl transfer step. Substrates withmultiple recognition sites do notshow burst kinetics and have turnover rate constants of 6h^-1. Catalytic turnover for themammalianenzyme is thus approximately 10-fold slower than that for the relatedbacterial enzymes. Our combinedresults show quantitatively that one enzyme is certainly capable ofboth maintenance and de novomethylation and that maintenance of the genomic methylation pattern ispreferred over the de novoestablishment of new patterns. Direct comparison of the mammalianenzyme with the bacterial DNAcytosine-C⁵ methyltransferase, M.SssI, indicatesdramatic differences in preferences for single-stranded,double-stranded, and hemimethylated double-stranded substrates.Moreover, the specificity hierarchy shownfor the M.SssI is derived from very different changes inK_m and catalysis than those observed forthemammalian DCMTase. These results demonstrate that theM.SssI, and perhaps other DNA cytosinemethyltransferases from bacteria, is functionally dissimilar to themammalian enzyme.