Genetics of telomerase in C. elegans

描述(申请人提供)：端粒酶逆转录端粒重复序列到染色体末端。该项目的长期目标是开发线虫作为研究端粒酶和端粒生物学的系统。线虫具有全着丝粒染色体，这有助于端粒酶缺乏时发生的初始端到端染色体融合事件的遗传分离。分离和分析来自不同遗传背景的端到端融合将为其起源提供前所未有的见解，并可能揭示一些与正常端粒相互作用的DNA损伤反应蛋白的意想不到的功能。基因筛查已经确定了端粒酶在酵母中端粒发挥作用所需的四种蛋白质，而我们的正向遗传筛查表明，线虫以及其他多细胞生物体中需要10种或更多的蛋白质。该领域的一个模型表明，端粒可能在端粒酶招募之前被感知为DNA双链断裂。然而，我们已经确定，促进DNA链间交联修复的MRT-1核酸酶是端粒酶作用于线虫端粒所必需的。因此，端粒酶可能通过一条能够对DNA链间交联物做出反应的途径被招募到端粒。我们怀疑这一假说可能与端粒进化有关，并计划使用遗传和生化方法来验证这一可能性。此外，我们还将鉴定和鉴定在体内导致线虫端粒酶活性缺失的四个新突变(279、222G、36F和3211E)。最后，将进行进一步的突变筛选，以努力饱和体内端粒酶活性所需的基因。端粒酶只在细胞周期中短暂地作用于端粒，因此线虫强大的遗传学可能有助于在多细胞生物体中确定体内负责端粒酶活性的一套基因。端粒酶的公共卫生相关性缺陷会导致致命的人类遗传性疾病，如再生障碍性贫血、先天性角化不良和肺纤维化：导致这些人类疾病的突变基因可能由这项研究项目定义。鉴于大多数癌症表达端粒酶，而大多数正常体细胞不表达端粒酶，我们的研究也可能有助于确定可能成为治疗多种癌症的有用靶点的蛋白质和/或途径。最后，该项目可能定义与端粒诱导的基因组不稳定相关的重组中间体和途径，这可能会促进肿瘤的发展。

DESCRIPTION (provided by applicant): Telomerase reverse transcribes telomere repeats onto chromosome termini. The long- term goal of this project is to develop the nematode C. elegans as a system for the study of telomerase and telomere biology. C. elegans possesses holocentric chromosomes that facilitate genetic isolation of initial end-to-end chromosome fusion events that occur when telomerase is deficient. Isolation and analysis of end-to-end fusions from various genetic backgrounds will provide unprecedented insight into their genesis and may reveal unexpected functions of some DNA damage response proteins that interact with normal telomeres. Genetic screens have identified four proteins that are required for telomerase to function at telomeres in yeast, whereas our forward genetic screens indicate that 10 or more proteins are required in C. elegans and perhaps in other multicellular organisms. One model in the field suggests that telomeres may be sensed as DNA double-strand breaks prior to recruitment of telomerase. However, we have determined that the MRT-1 nuclease, which facilitates DNA interstrand crosslink repair, is required for telomerase to act at C. elegans telomeres. Thus, telomerase may be recruited to telomeres via a pathway that can respond to DNA interstrand crosslinks. We suspect that this hypothesis may be relevant to telomere evolution and plan to test this possibility using genetic and biochemical approaches. In addition, we shall identify and characterize four new mutations that result in deficiency for telomerase activity in vivo in C. elegans (279, 222g, 36f and 3211e). Finally, further mutant screens will be performed in an effort to saturate for genes required for telomerase activity in vivo. Telomerase only acts at telomeres for a fleeting moment during the cell cycle, so the powerful genetics of C. elegans may help to identify the suite of genes responsible for telomerase activity in vivo in multicellular organisms. PUBLIC HEALTH RELEVANCE Deficiency for telomerase causes lethal human hereditary disorders such as aplastic anemia, dyskeritosis congenita and pulmonary fibrosis: genes that mutate to cause these human diseases may be defined by this research project. Given that most cancers express telomerase whereas most normal somatic cells do not, our studies may also help to define proteins and/or pathways that may be useful targets for treatment of a wide variety of cancers. Finally, this project may define recombination intermediates and pathways that are relevant to telomere-induced genome instability, which may promote tumor development.