Role of Nucleases in RNA Primer Removal and Mutagenesis

描述(由申请人提供)：癌症研究进展，从使用强大的模型系统的研究中受益匪浅，有力地支持了这样的假设，即导致基因组稳定性降低的突变积累是肿瘤发生的关键早期事件。在真核细胞DNA复制过程中，适当地实现Okazaki片段的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，DNA聚合酶α(Primase)合成了多个RNA引物和延伸的DNA片段。然而，与其他DNA聚合酶不同的是，该酶缺乏校对功能。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。这项建议旨在确定在酵母和哺乳动物细胞系统中核酸酶驱动的“阿尔法片段”处理或Okazaki片段成熟的详细分子机制。在过去的资助期间，我们已经确定了三种单独的核酸酶在这一过程中的作用，包括酿酒酵母核糖核酸酶H(35)、ScRad27或人fen-L和外切核酸酶-1，以及当这些核酸酶缺陷时的突变后果。目前的提议重点是检验一个中心假设，即两个相互作用的核酸酶复合体(DNA2-RPA和FEN-1-ROA1)顺序处理Okazaki片段的阿尔法片段。当Fen-1核酸酶活性被基因内建片段(如简单重复序列)抑制时，Werner综合征蛋白(WRN)和Fen-1核酸酶复合体采取另一种途径来解析Okazaki片段移位的α片段的内在二级结构。通过一系列强有力的系统分析，我们打算获得这三种核酸酶复合体在不同情况下如何共同作用于α片段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也将建立这种机制、独特的诱变表型(S)和遗传病发展之间的关系。

DESCRIPTION (provided by applicant): Cancer research progress, having benefited greatly from studies using powerful model systems, strongly supports the hypothesis that accumulation of mutations leading to decreased genome stability is a critical early event in tumorigenesis. 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 extended DNA-fragments are synthesized by DNA polymerase alpha (primase). However, this enzyme lacks 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 "alpha-segment" processing or for Okazaki fragment maturation in yeast and mammalian cell systems. For the last funding period, we have defined the roles of three individual nucleases in the process, including S. cerevisiae RNase H(35), ScRad27 or human FEN-l, and exonucleases-1, and mutagenic consequences when these nucleases are defective. The current proposal focuses to test a central hypothesis that two mutually interactive nuclease complexes (DNA2-RPA and FEN-1-ROA1) sequentially process the alpha-segment of the Okazaki fragment. When the FEN-1 nuclease activity is inhibited by genetically built-in blocks, such as simple repeat sequences, Werner syndrome protein (WRN) and FEN-1 nuclease complex takes an alternative route to resolve instrinsic secondary structure of the displaced alpha-segment of the Okazaki fragment. Through a series of vigorous systematic analyses, we intend to obtain a high resolution image of how these three nucleases complexes collectively work towards alpha-segment 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 also establish a relationship between this mechanism, unique mutagenic phenotype(s), and development of genetic diseases.