Molecular analyses of RECQ1 functions in genome maintenance

描述（由申请人提供）：RecQ解旋酶家族是一组高度保守的DNA解绕酶，在所有生命王国中保护基因组稳定性至关重要。人类的RecQ同源物包括RECQ1、BLM、WRN、RECQL4和RECQ5。虽然这五种人类RecQ蛋白在催化核心上是相似的，并且在体外具有一定的生化特性，但它们显然不是多余的。BLM、WRN和RecQ4突变分别与Bloom、Werner和rothmond - thomson综合征的不同遗传疾病相关。因此，一个RecQ蛋白的缺陷足以引起基因组的不稳定性，而其他RecQ同源物无法补偿这种不稳定性。然而，是什么让每个RecQ蛋白独一无二还不清楚。剖析每个人类RecQ解旋酶的功能，并比较它们之间的异同，将揭示DNA代谢中RecQ功能的哪些方面对基因组维持至关重要。本文的目的是研究RECQ1的分子功能，这是最丰富但最不具特征的人类RecQ解旋酶同源物。最近，我们已经证明RECQ1对基因组稳定性维持至关重要；它的缺乏会引起DNA损伤的积累和染色体的不稳定。RECQ1结合和解绕代表DNA重组修复中间体的DNA结构，并与参与调节基因重组的蛋白质在物理和功能上相互作用。此外，缺乏recq1的细胞对DNA损伤更敏感，并表现出自发升高的姐妹染色单体交换，使人联想到停滞的复制叉的异常修复。我们假设RECQ1通过其催化作用和与细胞蛋白伴侣的特定相互作用，在确保基因组稳定性方面起着关键作用。为了验证这一假设，我们建议对RECQ1的生化和细胞特性进行系统分析。我们将阐明RECQ1的生化活性如何使其在基因组稳定维持中实现其假定的功能：1)阐明RECQ1在基因组稳定维持的DNA修复途径中的作用；2)确定RECQ1中特定催化和细胞功能所必需的关键氨基酸残基；3)利用无偏生化方法鉴定RECQ1的新蛋白相互作用，并分析其功能意义。这些研究结果将对确定RECQ1的生物学作用具有重要意义。这将有助于解剖分子细节，解释人类RecQ解旋酶在基因组稳定维持途径中生物学功能的异同，以预防癌症和早衰。

DESCRIPTION (provided by applicant): The RecQ helicase family is a group of highly conserved DNA unwinding enzymes critical in guarding genome stability in all kingdoms of life. Human RecQ homologs include RECQ1, BLM, WRN, RECQL4, and RECQ5. Although the five human RecQ proteins are similar in their catalytic core and share certain biochemical properties in vitro, they are clearly not redundant. Mutations in BLM, WRN and RecQ4 are associated with distinct genetic disorders of Bloom, Werner, and Rothmund-Thomson syndromes, respectively. Thus, a defect in one RecQ protein is sufficient to cause genomic instability that cannot be compensated by other RecQ homologs. However, what makes each RecQ protein unique is not understood. Dissecting the functions of each human RecQ helicase, and comparing the similarities and differences among them, will reveal which aspects of RecQ functions in DNA metabolism are essential for genome maintenance. The goal of this proposal is to examine molecular functions of RECQ1, the most abundant but least characterized human RecQ helicase homolog. Recently, we have shown that RECQ1 is essential for genome stability maintenance; its deficiency induces accumulation of DNA damage and chromosomal instability. RECQ1 binds and unwinds DNA structures that represent intermediates of DNA recombination repair, and interacts physically and functionally with proteins involved in regulating genetic recombination. Moreover, RECQ1-deficient cells are more sensitive to DNA damage and display spontaneously elevated sister chromatid exchanges reminiscent of aberrant repair of stalled replication forks. We hypothesize that RECQ1 plays critical roles in ensuring genome stability by virtue of its catalytic actions and specific interactions with cellular protein partners. To test this hypothesis, we are proposing systematic analyses of the biochemical and cellular characteristics of RECQ1. We will elucidate how biochemical activities of RECQ1 allow it to achieve its putative functions in genome stability maintenance by: 1) elucidating role(s) of RECQ1 in DNA repair pathways of genome stability maintenance; 2) determining critical amino acid residues of RECQ1 essential for specific catalytic and cellular functions; and 3) identifying novel protein interactions of RECQ1 using unbiased biochemical approaches and investing their functional significance. Results from these studies will be important to establish biological roles of RECQ1. This should facilitate dissecting the molecular details that explain similarities and differences in the biological functions of human RecQ helicases in pathways of genome stability maintenance to prevent cancer and premature aging. PUBLIC HEALTH RELEVANCE: RECQ1 belongs to the RecQ family of DNA helicases members of which are associated with rare diseases of premature aging and cancer predisposition in humans. Thus, the functions of RecQ helicases have a direct impact on human health. The presence of multiple RecQ homologs in humans indicates functional specialization; elucidating the molecular function(s) of RECQ1 helicase should, therefore, provide important insights to the mechanisms of genome stability maintenance that prevent development of cancer and early onset of aging.