Biochemical basis of WRN and RecQ helicase function

WRN 和 RecQ 解旋酶功能的生化基础

• 批准号: 6810554
• 负责人: DAVID KEITH ORREN
• 金额: $ 28.26万
• 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/6810554
• 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解旋酶可以通过尚未知道的机制维持基因组稳定性。当它们的功能丢失时，非法重组的水平会大大增加。毫不奇怪，人类遗传性RECQ缺乏疾病（分别由WRN，BLM和RECQL4的缺陷引起的Werner，Bloom和Rothmund-Thomson综合症）表明早期发作和癌症的频率增加。重要的是，Werner综合征还显示出许多与年龄有关的问题的加速发展。尽管这些疾病具有独特的表型，但RECQ家族成员在保守的中央解旋酶结构域内和C终端保持高度同源性，这表明它们可能具有共同的机械功能和/或DNA底物特异性。我们将此扩展的序列保护称为RECQ扩展的核心。他们的非法重组表型表明，RECQ解旋酶在重组或抗重组途径中起作用，或者可能在解决复制叉堵塞的情况下起作用。我们的实验室最近在WRN和其他RECQ解旋酶中发现了与这些途径中假定角色一致的链配对和链交换活动。我们假设RECQ扩展的核心形成了一个功能单元，该功能单元包括DNA结合和催化活性。此外，我们建议RECQ扩展的核心的功能是协调DNA结合并放松，以实现复杂重组或复制中间体中的链交换反应。该假设符合RECQ成员的推定作用以及有关这些蛋白质的当前生化知识。 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.我们使用WRN的发现将与阐明其DNA代谢作用和癌变基础的特定机制和某些衰老表型高度相关。 RECQ解旋酶之间的序列保护还表明，我们的发现将适用于其他RECQ解旋酶（包括BLM和RECQL4）的功能及其与人类健康的关系。