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，引发酶（RNA聚合酶和DNA Pol的异源四聚体）合成多个RNA引物和立即邻接的DNA片段。然而，这两种酶都缺乏与其他DNA聚合酶不同的校对阅读功能。因此，该初始RNA-DNA片段（冈崎片段的α片段）是高度致突变的，并且必须由核酸酶复合物加工。本研究的目的是阐明真核生物细胞核和线粒体中核酸酶驱动的RNA引物加工的分子机制。在上一个资助期，我们已经确定了几种核酸酶在过程中的作用，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。我们还积累了确凿的证据证明，核酸酶解旋酶DNA 2专门定位于线粒体，并在线粒体DNA复制过程中的RNA引物去除中起着至关重要的作用。这些新的令人兴奋的观察促使我们在这次更新申请中开发新的和额外的具体目标。目前的建议集中于测试一个中心假设，即1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，并防止由于内在的DNA序列障碍和外源性的侮辱致突变应力。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，在酵母和哺乳动物细胞系统中的冈崎片段成熟的核酸酶驱动的“1-节段”加工，在复制正常的DNA序列和重复的DNA序列区域，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。本系统性研究提供的信息将确定该机制、独特的致突变表型与癌症发生之间的关系。

DESCRIPTION (provided by applicant): Appropriate implementation of Okazaki fragment maturation during DNA replication in eukaryotic cells is a fundamental mechanism for avoidance of mutations 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 of the enzymes lack a proof reading function, different from the other DNA polymerases. Therefore, this initial RNA-DNA fragment (alpha- segment of the Okazaki fragment) is highly mutagenic and has to be processed by nuclease complexes. This proposal aims to define detailed molecular mechanism for the nuclease-driven RNA primer processing in eukaryotic nuclei and mitochondria. For the last funding period, we have defined the roles of several nucleases in the processes, including S. cerevisiae RNase H(35), ScRad27 or human FEN1, and exonuclease-1, and mutagenic consequences when these nucleases are defective. We have also accumulated solid evidence to demonstrate that nuclease helicase DNA2 exclusively localizes into mitochondria, and plays a vital role in RNA primer removal during mitochondrial DNA replication. These novel exciting observations prompted us to develop new and additional specific aims in this renewal application. The current proposal focuses to test a central hypothesis that 1-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 "1-segment" processing in Okazaki fragment maturation in yeast and mammalian cell systems, 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. Information made available from this systematic study will establish a relationship between this mechanism, unique mutagenic phenotype(s), and development of cancers.