Functions of the BLM Helicase in Telomere Maintenance

摘要 人BLM编码recQ样DNA解旋酶，其对于维持基因组稳定性是重要的。 当这两个基因的拷贝都发生突变时，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易感性。来自BS个体的细胞的特征在于染色体 断裂和其他染色体异常，表明增加的体细胞重组。 值得注意的是，同源染色体之间的端粒缔合（TA）也存在于非同源染色体中。 来自BS个体的永生化和永生化细胞。在BLM基因的定位克隆之后， 本实验室研究了BLM解旋酶在DNA双链断裂修复中的功能 过程如非同源末端连接、同源重组介导的修复，以及 合成依赖性链退火。我们的工作也表明了BLM在重组中的作用- 端粒延长或ALT（端粒交替延长）介导的机制， 在没有端粒酶的情况下维持/延长端粒。BLM优先与端粒结合- 使用ALT在细胞中特异性结合蛋白TRF 1和TRF 2;其解旋酶活性可通过这些调节 交互.我们的初步数据确定和验证了其他蛋白质，独特的相互作用与BLM和 TRF 2在细胞中使用ALT，证明这些蛋白质相互作用修饰BLM的酶活性， 其配偶体拓扑异构酶II α，并显示五个假定磷酸化位点的修饰可以改变 DNA底物的解旋。我们假设BLM复合物的形成和修饰发生在 在保护端粒所需的特定核酸交易期间动态地进行比对， 同源端粒处的染色体序列，以允许链侵入和延伸，和/或 最终解开端粒。这些想法将通过对BLM改性的分析来研究， 定位和蛋白质伴侣在端粒延长，并通过修改这些相互作用或 使用基因工程小鼠进行体外和体内修饰。这个应用程序的直接目标 是确定BLM维持端粒的机制。这项工作具有重要意义 对了解细胞如何保持其基因组完整性，它们如何衰老或永生的影响，以及 最终用于开发更好的肿瘤治疗策略。项目叙述 易患癌症的遗传综合征为我们提供了一个研究基因和 这些蛋白质对于防止正常细胞成为肿瘤细胞非常重要。BLM解旋酶是一种 这些蛋白质，因为它似乎是维持人类基因组稳定所必需的。其作用 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，BLM的研究为我们提供了一个学习如何 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.