Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family
Domain mapping of Escherichia coli RecQ defines the roles of conserved N- and C-terminal regions in the RecQ family
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DOI：
10.1093/nar/gkg376
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发表时间：
2003-06-01
影响因子：
14.9
通讯作者：
Keck, JL
中科院分区：
文献类型：
--
作者：
Bernstein, DA;Keck, JL
RecQ DNA解旋酶在DNA复制、重组和修复中发挥作用。尽管这个酶家族在细胞中扮演的确切角色仍然难以捉摸，但RecQ蛋白的重要性是显而易见的；人类三个RecQ基因中的任何一个突变都会导致基因组不稳定和癌症。在这份报告中，蛋白水解法被用来定义大肠杆菌RecQ的两个结构域，揭示了一个大的(类似于59 kDa)N-末端和一个小的(类似于9 kDa的)C-末端结构域。一个短的N末端片段(7或21个残基)也被证明对蛋白酶敏感。去除这些RecQ区域的效果在体外被测试。去除RecQ中的21个N末端残基会严重降低其DNA依赖的ATPase和解旋酶的活性，但不影响其在电泳迁移率改变分析中与DNA的结合能力。相反，从RecQ中去除类似于9 kDa的C-末端结构域会产生一个具有正常水平的ATPase和解旋酶活性的片段，但这已经失去了与DNA稳定结合的能力。这些结果证实了N端序列基序在RecQ催化功能中的生化作用以及C端RecQ结构域在稳定的DNA结合中的作用。
RecQ DNA helicases function in DNA replication, recombination and repair. Although the precise cellular roles played by this family of enzymes remain elusive, the importance of RecQ proteins is clear; mutations in any of three human RecQ genes lead to genomic instability and cancer. In this report, proteolysis is used to define a two-domain structure for Escherichia coli RecQ, revealing a large (similar to59 kDa) N-terminal and a small (similar to9 kDa) C-terminal domain. A short N-terminal segment (7 or 21 residues) is also shown to be sensitive to proteases. The effects of removing these regions of RecQ are tested in vitro. Removing 21 N-terminal residues from RecQ severely diminishes its DNA-dependent ATPase and helicase activities, but does not affect its ability to bind DNA in electrophoretic mobility shift assays. In contrast, removing the similar to9 kDa C-terminal domain from RecQ results in a fragment with normal levels of ATPase and helicase activity, but that has lost the ability to stably associate with DNA. These results establish the biochemical roles of an N-terminal sequence motif in RecQ catalytic function and for the C-terminal RecQ domain in stable DNA binding.