Functions of the BLM Helicase in Telomere Maintenance

描述（由申请人提供）：人类BLM编码一种类似recq的DNA解旋酶，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，就会产生一种遗传性疾病，即布鲁姆综合征（BS），其特征是对太阳敏感、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都有极大的易感性。来自BS个体的细胞以染色体断裂和其他染色体异常为特征，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（TAs）也存在于来自BS个体的非永生化和永生化细胞中。继BLM基因的定位克隆之后，我们的实验室研究了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。我们的工作也提示了BLM在重组介导的端粒延伸或ALT（端粒的选择性延长）机制中的作用，这是在端粒酶缺失的情况下维持/延长端粒的过程。在使用ALT的细胞中，BLM优先与端粒特异性结合蛋白TRF1和TRF2结合；它的解旋酶活性可以通过这些相互作用来调节。我们的初步数据鉴定并验证了使用ALT在细胞中与BLM和TRF2相互作用的其他蛋白质，证明这些蛋白质相互作用改变了BLM及其伙伴拓扑异构酶iα的酶活性，并表明对五个假定的磷酸化位点的修饰可以改变DNA底物的解绕。我们假设BLM复合物的形成和修饰是在特定的核酸交易过程中动态发生的，这些交易需要保护端粒，在同源端粒上排列染色体序列，允许链入侵和延伸，和/或最终解开端粒。这些想法将通过分析端粒延伸过程中BLM修饰、定位和蛋白质配对，以及在体外和体内使用基因工程小鼠修饰这些相互作用或修饰来研究。本应用程序的直接目标是确定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，细胞如何衰老或不朽，并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变成肿瘤很重要。BLM解旋酶是这些蛋白质中的一种，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中的作用尤其重要，因为正是这些机制使细胞获得无限生长的能力。因此，对BLM的研究为我们提供了一个学习如何在治疗环境中控制癌细胞生长的机会。

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.