Biochemical basis of WRN and RecQ helicase function

描述(由申请人提供)：RecQ解旋酶通过一种未知的机制来维持基因组的稳定性。当它们的功能丧失时，非法重组的水平会显著增加。毫不奇怪，人类遗传性RecQ缺乏症(Werner、Bloom和Rothmund-Thomson综合征，分别由WRN、BLM和RECQL4缺陷引起)表现出早期发病和癌症发生率增加。重要的是，沃纳综合征也表现出许多与年龄相关的问题的加速发展。虽然这些疾病有不同的表型，但RecQ家族成员在保守的中央解旋酶结构域及其C端保持着高度的同源性，表明它们可能具有共同的机制功能和/或DNA底物特异性。我们将这种扩展的序列守恒称为RecQ扩展核心。它们的非法重组表型表明，RecQ解旋酶在重组或反重组途径中发挥作用，或者可能在解决复制分叉阻塞方面发挥作用。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可能扮演的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包含DNA结合和催化活性的功能单元。此外，我们认为，RecQ扩展核心的功能是协调DNA结合和解离，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成员的假定角色和关于这些蛋白质的当前生化知识。在这个建议中，WRN被用作一个模型RecQ解旋酶，用于1)表征链交换活性，反映链配对和解离活动之间的假定协调，2)检测复制和重组中间体的DNA结合性质和底物专一性，以及3)产生定位突变体，精确定位RecQ扩展核心及其单个结构域的DNA结合、酶和生理功能。我们对WRN的研究结果将与阐明其DNA代谢作用和导致癌症发生和某些衰老表型的特定机制高度相关。RecQ解旋酶之间的序列保守性也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。

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.