Role of Nucleases in RNA Primer Removal and Mutagenesis

描述（由申请人提供）：在真核细胞DNA复制过程中适当实施Okazaki片段成熟是避免突变和基因组稳定的基本机制。在滞后链DNA合成过程中，多个RNA引物和立即连接的DNA片段由引物酶（RNA聚合酶和DNA Pol的异四聚体）合成。然而，这两种酶都缺乏校对功能。因此，这个初始RNA-DNA片段（冈崎片段的α片段）是高度诱变的，必须由核酸酶复合物处理。本提案旨在定义真核生物细胞核和线粒体中核酸酶驱动的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，包括葡萄球菌RNase H（35）、ScRad27或人类FEN1和核酸外切酶-1，以及这些核酸酶缺陷时的致突变后果。母体提案继续我们的重点是验证一个中心假设，即a段加工是维持基因组完整性和防止由于内在DNA序列障碍和外源损伤引起的诱变胁迫的细胞机制的重要组成部分。缺乏这种整合机制可能导致高发生率的突变和致癌。我们将进一步定义细胞核和线粒体中正常DNA序列和重复DNA序列区域复制过程中冈崎片段成熟过程中核酸酶驱动的“a段”加工的详细分子机制。通过一系列有力的系统分析，我们打算获得这些核酸酶复合物如何在不同情况下共同作用于RNA引物加工的高分辨率图像，并将酵母和哺乳动物系统（包括人类细胞系和转基因小鼠）的体外和体内数据联系起来。最近，我们发现两种主要的核酸酶FEN1和DNA2定位于线粒体中，并协同处理复制和修复DNA中间体，以连接和完成环状mtDNA的复制和修复。这些新颖而令人兴奋的观察结果促使我们扩大了研究范围：1)敲除DNA2基因，以确定DNA2介导的RNA引物去除缺陷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；2)将人类线粒体疾病中发现的DNA2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、独特的线粒体致突变表型以及癌症和其他遗传疾病的病理机制之间的关系。提出的研究也可能为线粒体癌症和其他疾病患者的新治疗方案奠定良好的基础。此外，拟议的研究将创造1个博士后职位，保留一名研究生，并涵盖两名现有关键科学家的工作，从而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.