The Escherichia coli RecQ helicase functions as a monomer
The Escherichia coli RecQ helicase functions as a monomer
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DOI：
10.1074/jbc.m303581200
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发表时间：
2003-09-12
影响因子：
4.8
通讯作者：
Xi, XG
中科院分区：
文献类型：
--
作者：
Xu, HQ;Deprez, E;Xi, XG
RecQ 解旋酶属于高度保守的 DNA 解旋酶的重要家族，在染色体维持中发挥关键作用，其缺陷已被证明会导致人类多种疾病和癌症。在这项工作中，使用多种生物化学和生物物理技术确定了大肠杆菌 RecQ 解旋酶的构象和功能特性。获得的结果清楚地表明，大肠杆菌 RecQ 解旋酶在浓度高达 20 μM 的溶液中以及在 4 至 37 摄氏度之间的温度范围内呈单体。此外，这些特性不受 ATP 的存在（蛋白质的解旋和易位活性严格需要）或其不可水解类似物 5'-腺苷基-β,γ-亚胺二磷酸的影响。与结构特性一致，功能分析表明 DNA 解旋活性和单链 DNA 刺激的 ATP 酶比活性均独立于 RecQ 浓度。 RecQ 蛋白的 ATPase 缺陷突变体进一步证实了单体状态。当野生型RecQ解旋酶与ATP酶缺陷型突变体混合时，解旋速率没有变化，表明非蛋白质-蛋白质相互作用参与了解旋过程。总而言之，这些结果表明 RecQ 解旋酶作为单体发挥作用，并提供有关 RecQ 解旋酶的结构和功能特性的新数据，这可能有助于阐明其作用机制。
The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 muM and in a temperature range between 4 and 37 degreesC. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that non-protein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.