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首页> 外文期刊>Biochemistry >Allosteric activator domain of maintenance human DNA (Cytosine-5) methyltransferase and its role in methylation spreading.
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Allosteric activator domain of maintenance human DNA (Cytosine-5) methyltransferase and its role in methylation spreading.

机译：维持人类DNA（Cytosine-5）甲基转移酶的变构活化剂域及其在甲基化扩散中的作用。

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摘要

The human maintenance DNA (cytosine-5) methyltransferase (hDNMT1) consists of a large N-terminal regulatory domain fused to a catalytic C-terminal domain by randomly repeated Gly-Lys dipeptides. Several N-terminal deletion mutants of hDNMT1 were made, purified, and tested for substrate specificity. Deletion mutants lacking 121, 501, 540, or 580 amino acids from the N-terminus still functioned as DNA methyltransferases, methylated CG sequences, and preferred hemimethylated to unmethylated DNA, as did the full-length hDNMT1. Methylated DNA stimulated methylation spreading on unmethylated CpG sequences for the full-length and the 121 amino acid deletion hDNMT1 equally well but not for the mutants lacking 501, 540, or 580 amino acids, indicating the presence of an allosteric activation determinant between amino acids 121 and 501. Peptides from the N- and C-termini bound methylated DNA independently. Point mutation analysis within the allosteric region revealed that amino acids 284-287 (KKHR) were involved in methylated DNA-mediated allosteric activation. Allosteric activation was reduced in the double point mutant enzymes D25 (K284A and K285A) and D12 (H286A and R287A). Retinoblastoma gene product (Rb), a negative regulator of DNA methylation, bound to the allosteric site of hDNMT1 and inhibited methylation, suggesting Rb may regulate methylation spreading.
机译：人类维持性DNA（胞嘧啶5）甲基转移酶（hDNMT1）由通过随机重复的Gly-Lys二肽融合到催化C端结构域的大N端调节结构域组成。制备，纯化hDNMT1的几个N末端缺失突变体，并测试底物特异性。 N末端缺少121、501、540或580个氨基酸的缺失突变体仍可充当DNA甲基转移酶，甲基化CG序列，并且与全长hDNMT1一样，仍优选半甲基化为未甲基化的DNA。甲基化DNA刺激的甲基化在未甲基化CpG序列上的全长和121个氨基酸缺失hDNMT1的甲基化同样好，但对于缺少501、540或580个氨基酸的突变体却没有，这表明在氨基酸121之间存在变构活化决定簇501.来自N末端和C末端的肽独立地结合甲基化的DNA。变构区域内的点突变分析表明，氨基酸284-287（KKHR）与甲基化DNA介导的变构活化有关。双点突变酶D25（K284A和K285A）和D12（H286A和R287A）的变构活化降低。视网膜母细胞瘤基因产物（Rb）是DNA甲基化的负调节剂，与hDNMT1的变构位点结合并抑制了甲基化，提示Rb可能调节甲基化的扩散。

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