Mechanisms of Error Prone Repair of DNA Breaks

描述(由申请人提供)：致癌涉及多种基因变化，这些变化是由于DNA损伤修复错误导致的。存在几种高度容易出错的DNA断裂修复机制，它们可以作为突变的来源，并驱动各种癌症的形成和/或维持。因此，确定细胞如何以及何时依赖容易出错的机制来修复DNA断裂并评估与这些过程相关的基因改变的细胞和病理后果具有重大的公共卫生利益。这一建议的目的是阐明真核生物中这些容易出错的DNA断裂修复机制之一的分子机制，以及新发现的对这种重组反应至关重要的重组蛋白。串联重复序列之间的重组是一种进化保守的容易出错的机制，它通过产生序列缺失来修复DNA断裂。为了确定这个重组过程的遗传成分，我们用一种结合酵母遗传学和微阵列技术的方法筛选了有效修复直接重复序列两侧断裂所需的基因。这一筛选发现了两个新的重组基因，SLX4和SAW1，它们起到了从重组中间产物中去除3‘瓣的功能。3‘瓣的切除还依赖于结构特异的内切酶复合体Rad1/Rad10，并决定细胞对癌症化疗药物的耐受性、基因靶向、端粒完整性、氧化损伤的修复和衰老的抑制。为了破译容易出错的重组的生化和分子基础，我们计划定义SAW1在重组和重组相关生物过程中的生化特性，包括修复阻止正在进行的复制分叉进展的DNA损伤。我们将使用纯化的重组蛋白和3‘瓣DNA重建重组过程中的3’瓣去除过程。这些信息将有助于制定临床策略，以减少或消除与致癌有关的突变修复，并为改进癌症治疗方法铺平道路。

DESCRIPTION (provided by applicant): Carcinogenesis involves multiple genetic changes that result from errors in repair of DNA damage. Several highly error prone mechanisms for repair of DNA breaks exist which can serve as the source of mutations and drive formation and/or maintenance of a variety of cancers. It is thus of significant public health interest to define how and when cells rely on error prone mechanisms to repair DNA breaks and assess cellular and pathological consequences of genetic alterations associated with these processes. The goal of this proposal is to elucidate the molecular mechanisms of one of these error prone repair mechanisms of DNA breaks in eukaryotes and the newly identified recombination proteins crucial for such recombination reaction. Recombination between tandem repeat sequences is an evolutionary conserved error prone mechanism that repairs DNA breaks by producing sequence deletions. To define genetic component of this recombination process, we screened genes needed for efficient repair of breaks flanking direct repeats with an approach that combines yeast genetics and microarray technology. This screen uncovered two new recombination genes, SLX4 and SAW1, that function in removal of 3' flaps from recombination intermediates. Removal of a 3' flap also depends on the structure specific endonuclease complex, Rad1/Rad10, and dictates cellular tolerance to cancer chemotherapeutic agents, gene targeting, telomere integrity, repair of oxidative damage and suppression of aging. To decipher biochemical and molecular basis of error prone recombination, we plan to define the biochemical properties of Saw1 in recombination and recombination related biological processes including repair of DNA lesions blocking ongoing replication fork progression. We will reconstitute the 3' flap removal process during recombination using purified recombination proteins and a 3' flap DNA. The information will help devise clinical strategies to reduce or eliminate mutagenic repair germane to carcinogenesis and pave the way for improved cancer therapeutics.