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以HhaI甲基化酶和限制性内切酶作为检测d(GCGC)序列中B型至Z型DNA构象变化的探针。

HhaI methylase and restriction endonuclease as probes for B to Z DNA conformational changes in d(GCGC) sequences.

作者信息

Zacharias W, Larson J E, Kilpatrick M W, Wells R D

出版信息

Nucleic Acids Res. 1984 Oct 25;12(20):7677-92. doi: 10.1093/nar/12.20.7677.

DOI:10.1093/nar/12.20.7677
PMID:6093048
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC320193/
Abstract

The capacity of the modification methylase (MHhaI) and restriction endonuclease (HhaI) form Haemophilus haemolyticus to methylate and cleave, respectively, recognition sites which are in right-handed B or left-handed Z structures was determined in vitro. Plasmids containing tracts of (dC-dG) as well as numerous individual d(GCGC) sites distributed around the vector were studied. Negative supercoiling was used to convert the (dC-dG) tracts (approximately 30 bp in length) from a right-handed to a left-handed conformation. (Methyl-3H)-SAM was used to localize and quantitate modified d(GCGC) recognition sites, whereas cleavage by HhaI was used to detect unmethylated sites. In the left-handed Z-form, the (dC-dG) blocks were not methylated by MHhaI and not cleaved by HhaI. A two-dimensional gel analysis of a family of 33 topoisomers treated with MHhaI revealed that the lack of methylation in the (dC-dG) blocks was directly correlated to the supercoil-induced B to Z transition in these segments. These results are significant with respect to enzyme-DNA interactions in general and provide the basis for using HhaI and MHhaI as probes for different DNA structures and conformational transitions under physiological conditions.

摘要

体外测定了溶血嗜血杆菌的修饰甲基化酶（MHhaI）和限制性内切酶（HhaI）分别对处于右手B型或左手Z型结构中的识别位点进行甲基化和切割的能力。研究了含有（dC-dG）片段以及分布在载体周围的众多单个d（GCGC）位点的质粒。使用负超螺旋将（dC-dG）片段（长度约为30 bp）从右手构象转变为左手构象。（甲基-³H）-SAM用于定位和定量修饰的d（GCGC）识别位点，而HhaI的切割用于检测未甲基化的位点。在左手Z型中，（dC-dG）片段未被MHhaI甲基化，也未被HhaI切割。对用MHhaI处理的33个拓扑异构体家族进行的二维凝胶分析表明，（dC-dG）片段中甲基化的缺失与这些片段中超螺旋诱导的B型到Z型转变直接相关。这些结果对于一般的酶-DNA相互作用具有重要意义，并为在生理条件下使用HhaI和MHhaI作为不同DNA结构和构象转变的探针提供了基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6889/320193/9d46129733c8/nar00338-0079-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6889/320193/f123bf3b6a8b/nar00338-0077-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6889/320193/9d46129733c8/nar00338-0079-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6889/320193/f123bf3b6a8b/nar00338-0077-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6889/320193/9d46129733c8/nar00338-0079-a.jpg

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