Frederick C A, Quigley G J, van der Marel G A, van Boom J H, Wang A H, Rich A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1988 Nov 25;263(33):17872-9. doi: 10.2210/pdb4dnb/pdb.
Methylation of nucleic acid bases is known to prevent the cleavage of DNA by restriction endonucleases. The effect on the conformation of the DNA molecule itself and hence its interactions with other DNA binding proteins has been a subject of general interest. To help address this question, we have solved the crystal structure at 2.0 A of the methylated dodecamer, d(CGCGAm6ATTCGCG), which contains the EcoRI recognition sequence and have compared the conformation of the methylated molecule with that of its nonmethylated counterpart. This methylation produces a bulky hydrophobic patch on the floor of the major groove of B-DNA which plays an important role in the mechanism of inhibition of EcoRI restriction activity. However, with the exception of small perturbations in the immediate vicinity of the methyl groups, the structure is virtually unchanged. Given the lack of a conformational change upon methylation, we have extended this thesis of the recognition process to other types of restriction systems and found that different restriction enzymes seem to have their own characteristic protein-DNA interactions. The relative spatial orientations of methylation sites and cleavage sites must play a major role in ordering protein secondary structure elements as well as subunit-subunit interactions along the DNA strand.
已知核酸碱基的甲基化可防止限制性内切酶切割DNA。其对DNA分子自身构象以及与其他DNA结合蛋白相互作用的影响一直是人们普遍关注的课题。为了帮助解决这个问题，我们解析了甲基化十二聚体d（CGCGAm6ATTCGCG）在2.0埃分辨率下的晶体结构，该十二聚体包含EcoRI识别序列，并将甲基化分子的构象与其未甲基化对应物的构象进行了比较。这种甲基化在B-DNA大沟底部产生了一个庞大的疏水区域，该区域在抑制EcoRI限制活性的机制中起重要作用。然而，除了甲基附近的微小扰动外，结构几乎没有变化。鉴于甲基化时缺乏构象变化，我们将这一识别过程的理论扩展到其他类型的限制系统，发现不同的限制酶似乎有其自身独特的蛋白质-DNA相互作用。甲基化位点和切割位点的相对空间取向必定在沿DNA链排列蛋白质二级结构元件以及亚基-亚基相互作用方面起主要作用。
