Role of Nucleases in RNA Primer Removal and Mutagenesis

描述(申请人提供)：在真核细胞的DNA复制过程中，适当地实施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引物和直接相连的DNA片段被引物酶(RNA聚合酶和DNA Pol的异源四聚体)合成。然而，与其他DNA聚合酶不同的是，这两种酶都缺乏校对功能。因此，这个初始的RNA-DNA片段(冈崎片段的阿尔法片段)具有高度的突变性，必须由核酸酶复合体处理。本建议旨在确定真核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。在上一个资助阶段，我们已经确定了几种核酸酶在这些过程中的作用，包括酿酒酵母RNaseH(35)，ScRad27或人FEN1，以及外切核酸酶-1，以及当这些核酸酶缺陷时的突变后果。我们还积累了确凿的证据，证明核酸酶解旋酶DNA2仅定位于线粒体，在线粒体DNA复制过程中对RNA引物的去除起着至关重要的作用。这些新的令人兴奋的观察结果促使我们在这次续订申请中制定了新的和额外的具体目标。目前的提议集中于检验一个中心假设，即1-片段处理是维持基因组完整性和防止由于内在DNA序列障碍和外源侮辱而引起的突变应激的细胞机制的重要组成部分。这种整合机制的缺失可能导致诱变和致癌的高发。我们将进一步确定在酵母和哺乳动物细胞系统中，在复制正常DNA序列和重复DNA序列区域的过程中，在细胞核和线粒体中，核酶驱动的“1-片段”加工的详细分子机制。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体如何在不同情况下共同作用于RNA引物处理的高分辨率图像，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)关联体外和体内数据。这项系统性研究提供的信息将建立这种机制、独特的诱变表型(S)和癌症发展之间的关系。

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.