Flynn J, Azzam R, Reich N
Department of Chemistry, University of California, Santa Barbara 93106, USA.
J Mol Biol. 1998 May 29;279(1):101-16. doi: 10.1006/jmbi.1998.1761.
Mammalian DNA cytosine-C5 methyltransferase modifies the CpG dinucleotide in the context of many different genomic sequences. A rigorous DNA binding assay was developed for the murine enzyme and used to define how sequences flanking the CpG dinucleotide affect the stability of the enzyme:DNA complex. Oligonucleotides containing a single CpG site form reversible 1:1 complexes with the enzyme that are sequence-specific. A guanine/cytosine-rich 30 base-pair sequence, a mimic of the GC-box cis-element, bound threefold more tightly than an adenine/thymine-rich sequence, a mimic of the cyclic AMP responsive element. However, the binding discrimination between hemi- and unmethylated forms of these DNA substrates was small, as we previously observed at the K(m)DNA level (Biochemistry, 35, 7308-7315 (1996)). Single-stranded substrates are bound much more weakly than double-stranded DNA forms. An in vitro screening method was used to select for CpG flanking sequence preferences of the DNA methyltransferase from a large, divergent population of DNA substrates. After five iterative rounds of increasing selective pressure, guanosine/cytosine-rich sequences were abundant and contributed to binding stabilization for at least 12 base-pairs on either side of a central CpG. Our results suggest a read-out of sequence-dependent conformational features, such as helical flexibility, minor groove dimensions and critical phosphate orientation and mobility, rather than interactions with specific bases over the course of two complete helical turns. Thus, both studies reveal a preference for guanosine/cytosine deoxynucleotides flanking the cognate CpG. The enzyme specificity for similar sequences in the genome may contribute to the in vivo functions of this vital enzyme.
哺乳动物DNA胞嘧啶-C5甲基转移酶可在许多不同的基因组序列背景下修饰CpG二核苷酸。我们针对鼠类酶开发了一种严格的DNA结合测定法，并用于确定CpG二核苷酸侧翼序列如何影响酶与DNA复合物的稳定性。含有单个CpG位点的寡核苷酸与该酶形成可逆的1:1复合物，且具有序列特异性。一个富含鸟嘌呤/胞嘧啶的30碱基对序列，即GC框顺式元件的模拟物，其结合紧密程度是富含腺嘌呤/胸腺嘧啶序列（即环磷酸腺苷反应元件的模拟物）的三倍。然而，正如我们之前在K(m)DNA水平上所观察到的（《生物化学》，35卷，7308 - 7315页（1996年）），这些DNA底物的半甲基化和未甲基化形式之间的结合差异很小。单链底物的结合比双链DNA形式弱得多。我们使用一种体外筛选方法从大量不同的DNA底物群体中选择DNA甲基转移酶的CpG侧翼序列偏好性。经过五轮增加选择压力的迭代后，富含鸟苷/胞嘧啶的序列大量出现，并有助于在中央CpG两侧至少12个碱基对的结合稳定。我们的结果表明，读出的是序列依赖性的构象特征，如螺旋柔韧性、小沟尺寸以及关键磷酸基团的方向和移动性，而不是在两个完整螺旋圈过程中与特定碱基的相互作用。因此，两项研究都揭示了对同源CpG侧翼鸟苷/胞嘧啶脱氧核苷酸的偏好。该酶对基因组中相似序列的特异性可能有助于这种重要酶的体内功能。
