Mechanisms of Mammalian Double-Strand Break Repair

 描述（由申请人提供）：染色体重排形式的基因组不稳定性是癌细胞的标志，也是肿瘤发生的驱动因素。越来越多的证据表明，一种称为微同源介导的末端连接（MMEJ）的易出错的双链断裂修复替代形式通过利用序列微同源重组断裂的DNA来促进与DNA缺失相关的染色体重排。MMEJ不同于经典的非同源末端连接（NHEJ）途径，因为它以Ku和连接酶IV独立的方式发挥作用，因此被称为替代末端连接（alt-EJ）。虽然MMEJ似乎是alt-EJ的主要形式，但这种难以捉摸的途径的中心机制仍然未知。遗传学研究C.然而，线虫和果蝇的研究表明非典型A家族DNA聚合酶θ（Pol β）的中心作用。 在初步研究中，我们首次证明了由人POLQ表达的聚合酶结构域（本文中称为PollQ）执行含有具有两个或更多个同源性碱基对的3'单链DNA（ssDNA）突出端的DNA的MMEJ，包括在端粒之后建模的DNA。我们表明，MMEJ是特定的Pol？？，是促进相对突出端之间的氢键形成，并依赖于Pol？？在体内。值得注意的是，我们发现，Pol表现出DNA末端连接和微同源退火活动，其复制功能分开。然而，聚合酶利用相对的突出端作为反式模板来稳定DNA突触。我们进一步发现Pol优先对含有5 '末端磷酸的DNA进行MMEJ，这表明与参与NHEJ的X家族聚合酶在功能上相似。此外，我们确定了一个保守的插入环结构域的聚合酶，这是必不可少的MMEJ和高阶结构的聚合酶，这可能有利于DNA拴系。最后，我们提出的数据表明，Pol？具有末端转移酶活性，这被认为有助于MMEJ。 我们建议通过发展以下具体目标来进一步表征Pol的生化机制及其在MMEJ中的参与：1.阐明聚乙二醇促进MMEJ的作用机制; 2.研究并鉴定Poll末端转移酶活性; 3.为了表征全长Pol.总之，这些研究将为非典型A家族DNA聚合酶theta的活性提供新的见解，特别是其在双链断裂的MMEJ中的作用，因此对DNA修复研究界做出了重大贡献。

 DESCRIPTION (provided by applicant): Genome instability in the form of chromosome rearrangements is a hallmark of cancer cells and a driver of tumorigenesis. Mounting evidence indicates that an error-prone alternative form of double-strand break repair called microhomology-mediated end joining (MMEJ) promotes chromosome rearrangements associated with DNA deletions by utilizing sequence microhomology to recombine broken DNA. MMEJ is distinct from the classical non-homologous end joining (NHEJ) pathway since it functions in a Ku and Ligase IV independent manner and is therefore referred to as alternative end joining (alt-EJ). Although MMEJ appears to be the major form of alt-EJ, the central mechanism of this elusive pathway remains unknown. Genetic studies in C. elegans and Drosophila, however, suggest a central role for the atypical A-family DNA polymerase theta (Pol). In preliminary studies, we demonstrate for the first time that the polymerase domain expressed by human POLQ - herein referred to as Pol-performs MMEJ of DNA containing 3' single-strand DNA (ssDNA) overhangs with two or more base-pairs of homology, including DNA modeled after telomeres. We show that MMEJ is specific to Pol, is facilitated by hydrogen bond formation between opposing overhangs, and is dependent on Pol in vivo. Remarkably, we find that Pol exhibits DNA end joining and microhomology annealing activities separately from its replication function. Yet, the polymerase utilizes the opposing overhang as a template in trans to stabilize the DNA synapse. We further find that Pol preferentially performs MMEJ of DNA containing a 5'-terminal phosphate, which demonstrates a functional similarity to X-family polymerases involved in NHEJ. Additionally, we identify a conserved insertion loop domain in Pol that is essential for MMEJ and higher-order structures of the polymerase which likely facilitate DNA tethering. Lastly, we present data suggesting that Pol exhibits terminal transferase activity, which is thought to contribute to MMEJ. We propose to further characterize the biochemical mechanisms of Poland its involvement in MMEJ by developing the following specific aims: 1. To elucidate the mechanism of MMEJ promoted by Pol; 2. To investigate and characterize terminal transferase activity of Pol; 3. To characterize the activities of full-lenth Pol . In summary, these studies will provide new insight into the activities of the atypical A-family DNA polymerase theta, in particular its role in MMEJ of double-strand breaks, and therefore significantly contribute to the DNA repair research community.