Functions of the BLM Helicase in Telomere Maintenance

摘要 人类BLM编码一种类似RecQ的DNA解旋酶，它对维持基因组的稳定性很重要。 当这种基因的两个拷贝都发生突变时，就会导致遗传性疾病，称为布鲁姆综合征 (Bs)，特点是对阳光敏感，身材矮小，免疫缺陷，男性不育，以及巨大的 所有部位和类型的癌症的易感性。来自BS个体的细胞具有染色体特征 断裂和其他染色体异常，表明体细胞重组增加。 值得注意的是，同源染色体之间的端粒关联(TA)也存在于非 来自BS个体的永生化细胞和永生化细胞。在BLM基因的定位克隆之后， 本实验室研究了BLM解旋酶在DNA双链断裂修复中的作用 过程如非同源末端连接、同源重组介导的修复和 依赖于合成的链退火法。我们的工作还表明，BLM在重组中扮演了一个角色- 端粒延长或ALT(端粒交替延长)的中介机制 在没有端粒酶的情况下维持/延长端粒。BLM优先与端粒结合- 细胞中使用ALT的特异性结合蛋白TRF1和TRF2；其解旋酶活性可由这些调节 互动。我们的初步数据确定并验证了其他与BLM和BLM独特相互作用的蛋白质 在使用ALT的细胞中的TRF2，证明了这些蛋白质相互作用改变了BLM的酶活性和 它伙伴拓扑异构酶IIpha，并表明五个假定的磷酸化位点的修饰可以改变 脱氧核糖核酸底物的展开。我们假设BLM复合体的形成和修饰发生了 在保护端粒所需的特定核酸交易期间动态地对齐 同源端粒上的染色体序列，以允许链侵袭和延伸，和/或 最终解开端粒。这些想法将通过对BLM修改的分析来研究， 在端粒延长过程中的定位和蛋白质配对，并通过改变这些相互作用或 利用基因工程小鼠在体外和体内进行修改。这个应用程序的直接目标是 是为了确定BLM维持端粒的机制。这项工作具有重要的意义 对了解细胞如何保持其基因组完整性、它们如何衰老或变得永生的启示，以及 最终用于开发更好的肿瘤学治疗策略。项目叙事 易患癌症的遗传综合征为我们提供了一个研究基因和 对防止正常细胞发生肿瘤非常重要的蛋白质。博莱姆解旋酶是一种 这些蛋白质似乎是维持人类基因组稳定所必需的。它在世界经济中的作用 染色体末端的维持尤其重要，因为正是这些机制使细胞能够获得 无限增长的能力。因此，对BLM的研究为我们提供了一个机会，让我们了解如何 可以在治疗环境中控制癌细胞的生长。

ABSTRACT 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. PROJECT NARRATIVE 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.