Mechanisms of Error Prone Repair of DNA Breaks

描述（由申请人提供）：癌变涉及DNA损伤修复错误导致的多种遗传变化。DNA断裂的修复存在几个高度容易出错的机制，这些机制可以作为突变的来源，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遗传改变的细胞和病理后果，具有重大的公共卫生利益。本研究的目的是阐明真核生物DNA断裂的一种错误修复机制的分子机制，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易出错机制，它通过产生序列缺失来修复DNA断裂。为了确定这种重组过程的遗传成分，我们使用酵母遗传学和微阵列技术相结合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。该筛选发现了两个新的重组基因，SLX4和SAW1，它们的功能是去除重组中间体的3' flap。去除3'瓣也取决于结构特异性内切酶复合物Rad1/Rad10，并决定细胞对癌症化疗药物、基因靶向、端粒完整性、氧化损伤修复和衰老抑制的耐受性。为了破译易出错重组的生化和分子基础，我们计划定义Saw1在重组和重组相关的生物过程中的生化特性，包括修复DNA损伤，阻止正在进行的复制叉进展。我们将在重组过程中使用纯化的重组蛋白和3‘瓣DNA重建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.