Mechanisms of Mammalian Double-Strand Break Repair

 描述(申请人提供)：染色体重排形式的基因组不稳定是癌细胞的一个标志，也是肿瘤发生的驱动因素。越来越多的证据表明，一种容易出错的双链断裂修复的替代形式，称为微同源介导的末端连接(MMEJ)，通过利用序列微同源性重组断裂的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Ligase 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，但这一难以捉摸的途径的中心机制仍不清楚。然而，对线虫和果蝇的遗传学研究表明，非典型的A家族dna聚合酶theta(PolDNA聚合酶)起着中心作用。在初步研究中，我们首次证明了人类POLQ表达的聚合酶结构域--这里称为PolDNA--执行包含3‘单链(单链DNA)与两个或更多同源碱基对的DNA的MMEJ，包括模仿端粒的DNA。我们发现MMEJ是POL特异性的，通过相对悬垂之间氢键的形成而促进，并在体内依赖于POL。值得注意的是，我们发现POL除了具有复制功能外，还具有末端连接和微同源的退火活性。然而，聚合酶利用相反的突出物作为反式DNA突触的模板来稳定DNA突触。我们进一步发现，POL优先执行含有5‘-末端磷酸的DNA的MMEJ，这表明功能上与NHEJ中涉及的X-家族聚合酶相似。此外，我们在POL中发现了一个保守的插入环区，它对于MMEJ和聚合酶的高阶结构是必不可少的，这可能有助于DNA拴系。最后，我们提供的数据表明，POL具有末端转移酶活性，这被认为与MMEJ有关。我们建议通过以下具体目标来进一步研究POL的生化机制及其参与MMEJ：1.阐明POL促进MMEJ的机制；2.研究POL的末端转移酶活性；3.表征全长POL的活性。综上所述，这些研究将为非典型A家族DNA聚合酶theta的活动提供新的见解，特别是它在双链断裂的MMEJ中的作用，因此对DNA修复研究社区有重要贡献。

 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.