Functions of the BLM Helicase in Telomere Maintenance

描述（由申请人提供）：人BLM编码一种recQ样DNA解旋酶，其对维持基因组稳定性很重要。当该基因的两个拷贝都发生突变时，所产生的遗传性疾病，称为布卢姆综合征（BS），其特征是阳光敏感性，身材矮小，免疫缺陷，男性不育，以及对所有部位和类型的癌症的巨大易感性。来自BS个体的细胞的特征在于染色体断裂和指示增加的体细胞重组的其他染色体异常。值得注意的是，同源染色体之间的端粒缔合（TA）也存在于来自BS个体的非永生化和永生化细胞中。继BLM基因的定位克隆，我们的实验室已经研究了BLM解旋酶在DNA双链断裂修复过程中的功能，如非同源末端连接，同源重组介导的修复，和合成依赖的链退火。我们的工作还表明了BLM在端粒延长或ALT（端粒的交替延长）的重组介导机制中的作用，所述端粒延长或ALT是在不存在端粒酶的情况下维持/延长端粒的过程。BLM使用ALT优先与细胞中的端粒特异性结合蛋白TRF 1和TRF 2缔合;其解旋酶活性可以通过这些相互作用来调节。我们的初步数据确定和验证其他蛋白质，独特的相互作用与BLM和TRF 2在细胞中使用ALT，表明这些蛋白质的相互作用修改的酶活性的BLM和它的伴侣拓扑异构酶IIalpha，并表明，修改5个假定的磷酸化位点可以改变解旋的DNA底物。我们假设，BLM复合物的形成和修饰动态发生在特定的核酸交易，需要保护端粒，在同源端粒染色体序列对齐，允许链入侵和伸长，和/或最终解开端粒。这些想法将通过分析端粒延长过程中的BLM修饰，定位和蛋白质伴侣，并通过使用基因工程小鼠在体外和体内修改这些相互作用或修改来研究。本申请的直接目标是确定BLM维持端粒功能的机制。这项工作对于了解细胞如何保持其基因组完整性，它们如何衰老或变得不朽，以及最终开发更好的肿瘤治疗策略具有重要意义。公共卫生相关性：易患癌症的遗传综合征为我们提供了一个研究基因和蛋白质的机会，这些基因和蛋白质对防止正常细胞成为肿瘤很重要。BLM解旋酶是这些蛋白质之一，因为它似乎是维持人类基因组稳定性所必需的。它在维持染色体末端方面的作用尤其重要，因为正是这些机制使细胞能够获得无限生长的能力。因此，BLM的研究为我们提供了一个机会，让我们了解如何在治疗环境中控制癌细胞的生长。

DESCRIPTION (provided by applicant): Human BLM encodes a recQ-like DNA helicase that is important for the maintenance of genomic stability. When both copies of this gene are mutated, the resulting hereditary disease, known as Bloom's syndrome (BS), is characterized by sun-sensitivity, small stature, immunodeficiency, male infertility, and a tremendous predisposition to cancer of all sites and types. Cells from BS individuals are characterized by chromosome breakage and other chromosomal anomalies that are indicative of increased somatic recombination. Notably, telomeric associations (TAs) between homologous chromosomes are also present in non- immortalized and immortalized cells from BS individuals. Following the positional cloning of the BLM gene, our laboratory has investigated the functions of the BLM helicase in DNA double strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, and synthesis-dependent strand annealing. Our work has also suggested a role for BLM in recombination- mediated mechanisms of telomere elongation or ALT (alternative lengthening of telomeres), processes that maintain/elongate telomeres in the absence of telomerase. BLM preferentially associates with the telomere- specific binding proteins TRF1 and TRF2 in cells using ALT; its helicase activity can be modulated by these interactions. Our preliminary data identify and validate other proteins that uniquely interact with BLM and TRF2 in cells using ALT, demonstrate that these protein interactions modify enzymatic activity of BLM and its partner topoisomerase IIalpha, and show that modification of five putative phosphorylation sites can alter unwinding of DNA substrates. We hypothesize that BLM complex formation and modification occur dynamically during the specific nucleic acid transactions that are required to protect the telomere, to align chromosome sequences at homologous telomeres, to permit strand invasion and elongation, and/or ultimately to disentangle telomeres. These ideas will be investigated by analyses of BLM modification, localization and protein partnering during telomere elongation, and by modifying these interactions or modifications in vitro and in vivo using genetically engineered mice. The immediate goal of this application is to determine the mechanism by which BLM functions to maintain telomeres. This work has important implications for learning how cells maintain their genomic integrity, how they age or become immortal, and ultimately for developing better therapeutic strategies in oncology. PUBLIC HEALTH RELEVANCE: Inherited syndromes that predispose to cancer have provided us an opportunity to study the genes and proteins that are important for keeping normal cells from becoming neoplastic. The BLM helicase is one of these proteins, as it seems to be required to maintain stability of the human genome. Its role in the maintenance of chromosome ends is especially important, as it is these mechanisms that enable cells to gain the ability to grow indefinitely. The study of BLM therefore represents an opportunity for us to learn how we can control the growth of cancer cells in a therapeutic setting.