Role of Nucleases in RNA Primer Removal and Mutagenesis

描述（由申请人提供）：癌症研究进展极大地受益于使用强大模型系统的研究，强烈支持导致基因组稳定性降低的突变积累是肿瘤发生中的关键早期事件的假设。在真核细胞中DNA复制过程中适当地实现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引物和延伸的DNA片段由DNA聚合酶α（引物酶）合成。然而，这种酶缺乏证明阅读功能，不同于其他DNA聚合酶。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，并且必须由核酸酶复合物加工。该建议旨在定义酵母和哺乳动物细胞系统中核酸酶驱动的“α片段”加工或冈崎片段成熟的详细分子机制。在最后一个资助期，我们已经确定了三个单独的核酸酶在这个过程中的作用，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。目前的建议集中于测试一个中心假设，即两个相互作用的核酸酶复合物（DNA 2-RPA和FEN-1-ROA 1）顺序处理冈崎片段的α片段。当FEN-1核酸酶活性被遗传学上内置的阻断物（例如简单重复序列）抑制时，沃纳综合征蛋白（WRN）和FEN-1核酸酶复合物采取替代途径来解析冈崎片段的置换α片段的固有二级结构。通过一系列有力的系统分析，我们打算获得一个高分辨率的图像，这三个核酸酶复合物如何共同努力，在不同的情况下对α-段处理，并涉及在体外和体内数据使用酵母和哺乳动物系统，包括人类细胞系和转基因小鼠。本系统性研究提供的信息还将确立该机制、独特的致突变表型与遗传疾病发生之间的关系。

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.