Biochemical basis of WRN and RecQ helicase function

WRN 和 RecQ 解旋酶功能的生化基础

• 批准号: 6935883
• 负责人: DAVID KEITH ORREN
• 金额: $ 24.16万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-10 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6935883
• 关键词: DNA binding protein; DNA repair; DNA replication; DNA replication origin; SDS polyacrylamide gel electrophoresis; Werner' s syndrome; enzyme mechanism; enzyme structure; gene mutation; genetic recombination; helicase; nucleic acid metabolism; site directed mutagenesis

DESCRIPTION (provided by applicant): RecQ helicases act to maintain genomic stability by an as yet unknown mechanism. When their function is lost, levels of illegitimate recombination increase significantly. Not surprisingly, the human hereditary RecQ deficiency diseases (Werner, Bloom, and Rothmund-Thomson syndromes, caused by defects in WRN, BLM, and RECQL4, respectively) demonstrate early onset and increased frequency of cancer. Importantly, Werner syndrome also shows accelerated development of many age-related problems. Although these diseases have distinct phenotypes, RecQ family members maintain a high degree of homology within and C terminal to the conserved central helicase domain, suggesting that they may have a common mechanistic function and/or DNA substrate specificity. We refer to this extended sequence conservation as the RecQ expanded core. Their illegitimate recombination phenotypes suggest that RecQ helicases function in recombination or anti-recombination pathways, or possibly in resolution of replication fork blockage. Our laboratory has recently uncovered strand pairing and strand exchange activities in WRN and other RecQ helicases consistent with putative roles in these pathways. We hypothesize that the RecQ expanded core forms a functional unit that encompasses DNA binding and catalytic activities. Further, we propose that the function of the RecQ expanded core is to coordinate DNA binding and unwinding to achieve strand exchange reactions in complex recombination or replication intermediates. This hypothesis fits with the putative roles for RecQ members and current biochemical knowledge regarding these proteins. In this proposal, WRN is used as a model RecQ helicase for 1) characterizing strand exchange activity reflecting putative coordination between strand pairing and unwinding activities, 2) examining DNA binding properties and substrate specificity for replication and recombination intermediates, and 3) generating site-directed mutants that pinpoint the DNA binding, enzymatic, and physiological functions of the RecQ expanded core and its individual domains. Our findings with WRN will be highly relevant to elucidating its DNA metabolic role and specific mechanisms underlying carcinogenesis and certain aging phenotypes. Sequence conservation between RecQ helicases also indicates that our findings will be applicable to the functions of other RecQ helicases (including BLM and RECQL4) and their relationships to human health.

描述（由申请人提供）：RecQ解旋酶通过迄今未知的机制发挥维持基因组稳定性的作用。当它们的功能丧失时，非法重组的水平就会显着增加。毫不奇怪，人类遗传性 RecQ 缺陷性疾病（Werner、Bloom 和 Rothmund-Thomson 综合征，分别由 WRN、BLM 和 RECQL4 缺陷引起）表现出癌症的早发和发病率增加。重要的是，维尔纳综合征还显示出许多与年龄相关的问题的加速发展。尽管这些疾病具有不同的表型，但 RecQ 家族成员在保守的中央解旋酶结构域内和 C 端保持高度同源性，表明它们可能具有共同的机制功能和/或 DNA 底物特异性。我们将这种扩展序列保守称为 RecQ 扩展核心。它们的非法重组表型表明 RecQ 解旋酶在重组或抗重组途径中发挥作用，或者可能在解决复制叉阻塞中发挥作用。我们的实验室最近发现了 WRN 和其他 RecQ 解旋酶中的链配对和链交换活性，与这些途径中的假定作用一致。我们假设 RecQ 扩展核心形成一个包含 DNA 结合和催化活性的功能单元。此外，我们提出 RecQ 扩展核心的功能是协调 DNA 结合和解旋，以实现复杂重组或复制中间体中的链交换反应。这一假设符合 RecQ 成员的假定角色以及当前有关这些蛋白质的生化知识。在本提案中，WRN 被用作 RecQ 解旋酶模型，用于 1）表征链交换活性，反映链配对和解旋活性之间的假定协调，2）检查复制和重组中间体的 DNA 结合特性和底物特异性，以及 3）生成位点定向突变体精确定位 RecQ 扩展核心及其各个结构域的 DNA 结合、酶促和生理功能。我们对 WRN 的研究结果将与阐明其 DNA 代谢作用以及致癌和某些衰老表型的具体机制高度相关。 RecQ 解旋酶之间的序列保守性也表明我们的研究结果将适用于其他 RecQ 解旋酶（包括 BLM 和 RECQL4）的功能及其与人类健康的关系。