Role of Nucleases in RNA Primer Removal and Mutagenesis

描述（由申请人提供）：在真核细胞中DNA复制期间适当实施冈崎片段成熟是避免突变和基因组稳定性的基本机制。在滞后链DNA合成期间，多个RNA引物和直接邻接的DNA片段由引物酶（RNA聚合酶和DNA Pol的异源四聚体）合成。然而，这两种酶都缺乏校对阅读功能。因此，该初始RNA-DNA片段（冈崎片段的α片段）具有高度致突变性，必须由核酸酶复合物加工。本论文的主要目的是阐明真核生物细胞核和胞体中核酸酶驱动的RNA引物加工的详细分子机制。在上一个资助期，我们已经确定了几种核酸酶在这些过程中的作用，包括S。酿酒酵母RNA酶H（35）、ScRad 27或人FEN 1和外切核酸酶-1，以及当这些核酸酶有缺陷时的诱变后果。母公司的建议继续我们的重点是测试一个中心的假设，即a-段加工是细胞机制的重要组成部分，以保持基因组的完整性，并防止诱变应力由于内在的DNA序列障碍和外源性的侮辱。这种整合机制的缺陷可能导致高发生率的诱变和致癌作用。我们将进一步定义详细的分子机制的核酸酶驱动的“a-节段”加工冈崎片段成熟过程中的正常DNA序列和重复的DNA序列区域的复制，在细胞核中，以及在细胞核中。通过一系列强有力的系统分析，我们打算获得这些核酸酶复合物如何在不同的情况下共同工作对RNA引物加工的高分辨率图像，并使用酵母和哺乳动物系统，包括人类细胞系和转基因小鼠的体外和体内数据。最近，我们发现两个主要的核酸酶，FEN 1和DNA 2，定位于线粒体和合作过程复制和修复DNA中间连接和完成环状mtDNA复制和修复。这些新的和令人兴奋的观察促使我们扩大我们的范围：i）敲除DNA 2基因，以确定缺陷的DNA 2介导的RNA引物去除是否会导致线粒体基因组不稳定，从而促进癌症和其他遗传疾病，ii）将人类基于线粒体的疾病中鉴定的DNA 2突变的功能缺陷与病理机制联系起来。从这些额外的研究中获得的信息将建立这些新的线粒体基因的功能，独特的线粒体诱变表型，以及癌症和其他遗传疾病的病理机制之间的关系。这项拟议中的研究也可能为基于癌症和其他疾病的患者的新治疗方案奠定良好的基础。此外，拟议的研究将通过创建1个博士后职位和保留一名研究生并涵盖两名关键现有科学家的努力来产生直接的经济影响。 公共卫生相关性：拟议的研究将建立这些新的线粒体基因的功能之间的关系，独特的线粒体诱变表型，以及癌症和其他遗传疾病的病理机制，并可能为基于线粒体的癌症和其他疾病的患者提供新的治疗方案。此外，拟议的研究将通过创建1个博士后职位和保留一名研究生并涵盖两名关键现有科学家的努力来产生直接的经济影响。

DESCRIPTION (provided by applicant): Appropriate implementation of Okazaki fragment maturation during DNA replication in eukaryotic cells is a fundamental mechanism for mutation avoidance and genome stability. During lagging strand DNA synthesis, multiple RNA primers and immediately adjoined DNA-fragments are synthesized by primase (a hetero tetramer of a RNA polymerase and DNA Pol ). However, both enzymes lack a proof reading function. Therefore, this initial RNA-DNA fragment (alpha-segment of the Okazaki fragment) is highly mutagenic and must be processed by nuclease complexes. The parent proposal aims to define detailed molecular mechanism for the nuclease-driven RNA primer processing in eukaryotic nucleus and mitochondrion. For the last funding period, we have defined the roles of several nucleases in these processes, including S. cerevisiae RNase H (35), ScRad27 or human FEN1, and exonuclease-1, and mutagenic consequences when these nucleases are defective. The parent proposal continues our focus to test a central hypothesis that a-segment processing is a vital part of cellular mechanisms to maintain genomic integrity and prevent mutagenic stresses due to intrinsic DNA sequence obstacles and exogenous insults. Deficiency of this integrative machinery could lead to a high incidence of mutagenesis and carcinogenesis. We will further define detailed molecular mechanisms for the nuclease-driven "a-segment" processing in Okazaki fragment maturation during replication of normal DNA sequence and repetitive DNA sequence regions, in the nucleus as well as the mitochondrion. Through a series of vigorous systematic analyses, we intend to obtain a high resolution image of how these nuclease complexes collectively work towards RNA primer processing in different scenarios and to relate in vitro and in vivo data using yeast and mammalian systems, including human cell lines and transgenic mice. Recently, we found that two major nucleases, FEN1 and DNA2, are localized into mitochondria and cooperatively process replication and repair DNA intermediates for ligation and completion of circular mtDNA replication and repair. These novel and exciting observations prompted us to expand our scope: i) to knock out the DNA2 gene to determine if defective DNA2-mediated RNA primer removal causes mitochondrial genomic instabilities and consequently promotes cancers and other genetic diseases and ii) to link functional defects of the DNA2 mutations identified in human mitochondrion-based diseases to pathologic mechanisms. Information made available from these additional studies will establish a relationship among the functions of these novel mitochondrial genes, unique mitochondrial mutagenic phenotype(s), and pathological mechanisms of cancers and other genetic diseases. The proposed study may also set a good foundation for new treatment regimens to patients with mitochondrion-based cancers and other disorders. Moreover, the proposed research will have immediate economic impact by creating 1 postdoctoral position and retaining a graduate student and covering efforts of two key existing scientists. PUBLIC HEALTH RELEVANCE: The proposed studies will establish a relationship among the functions of these novel mitochondrial genes, unique mitochondrial mutagenic phenotype(s), and pathological mechanisms of cancers and other genetic diseases and may provide new treatment regimens to patients with mitochondrion-based cancers and other disorders. Moreover, the proposed research will have immediate economic impact by creating 1 postdoctoral position and retaining a graduate student and covering efforts of two key existing scientists.