Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences
Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences
复制标题
DOI：
10.1074/jbc.m802502200
复制
发表时间：
2008-07-18
影响因子：
4.8
通讯作者：
Reich, Norbert O.
中科院分区：
文献类型：
--
作者：
Coffin, Stephanie R.;Reich, Norbert O.
大肠杆菌DNA腺嘌呤甲基转移酶（EcoDam）可使5 '-GATC-3'序列中腺嘌呤的N-6位甲基化，在基因调控、错配修复和DNA复制中起重要作用。目前尚不清楚参与复制和基因表达调节的少数关键GATC位点如何发生差异甲基化，而对错配修复重要且分散在整个基因组中的类似20，000个GATC位点则发生广泛甲基化。我们先前的工作，仅限于pap调节子，表明甲基化效率是由直接侧翼的GATC位点序列控制的。我们将这些研究扩展到包括参与不同基因调控和DNA复制途径的GATC位点，以及先前显示在体内发生差异甲基化但其功能仍有待分配的位点。EcoDam没有显示出与来自这些来源的侧翼序列的变化的亲和力的变化，但甲基化动力学变化12倍。紧邻GATC位点的A束对甲基化动力学的这些差异有显著贡献。有趣的是，只有当聚腺苷酸位于GATC的5'时，才显示甲基化动力学的变化。当两个GATC位点位于同一DNA分子上时，优先甲基化被掩盖，除非两个位点都被大量非特异性DNA包围。因此，促进扩散和序列直接侧翼的目标位点有助于更高的顺序特异性EcoDam，我们建议，酶的不同生物学作用的一部分是由这两个因素，这可能是重要的其他酶，序列特异性修饰DNA。
Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the similar to 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.