The RecBCD enzyme initiation complex for DNA unwinding: Enzyme positioning and DNA opening
The RecBCD enzyme initiation complex for DNA unwinding: Enzyme positioning and DNA opening
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DOI：
10.1006/jmbi.1997.1259
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发表时间：
1997-10-10
影响因子：
5.6
通讯作者：
Smith, GR
中科院分区：
文献类型：
--
作者：
Farah, JA;Smith, GR
大肠杆菌 RecBCD 酶将 DNA 从游离双链 DNA 末端解旋，产生同源重组的单链 DNA 中间体。在不存在 ATP 的情况下，RecBCD 与游离 DNA 末端结合，形成 DNA 解旋的起始复合物。我们研究了这些由平端 DNA、5' 延伸和 3' 延伸 DNA 形成的复合物的结构。对单链 DNA 特异性试剂 KMnO4 和硫酸二甲酯的反应表明 RecBCD 以 Mg2+ 依赖性方式打开每个底物中末端的 5 个或 6 个碱基对。距 5' 末端 4 至 6 个核苷酸的胸腺嘧啶残基仅与 KMnO4 部分反应，表明部分 5' 末端链被酶部分屏蔽。 DNase I 足迹表明该酶相对于两条链中较长链的末端定位自身，但也注意到了一个例外。这些结果表明 RecBCD 打开 DNA 并定位自身以在具有不同类型末端的 DNA 上解旋的能力具有灵活性。它们还暗示了与不同类型末端结合的 RecBCD 酶的构象差异；这些构象差异可能与解旋周期中发生的构象差异有关。 (C) 1997 学术出版社有限公司。
The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. Ln the absence of ATP RecBCD binds to a free DNA end to form an initiation complex for DNA unwinding. We studied the structure of these complexes formed with blunt-ended, 5'-extended, and 3'-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg2+-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5' end were only partially reactive to KMnO4, suggesting that part of the 5'-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle. (C) 1997 Academic Press Limited.