Mechanisms of Error Prone Repair of DNA Breaks

描述（由申请人提供）：致癌作用涉及DNA损伤修复错误导致的多种遗传变化。存在几种用于修复DNA断裂的高度易错机制，其可充当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于错误倾向机制来修复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意义。本提案的目标是阐明真核生物中DNA断裂的这些错误倾向性修复机制之一的分子机制以及新鉴定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复是一种进化上保守的易错机制，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组成部分，我们筛选出的方法，结合酵母遗传学和微阵列技术的断裂侧翼直接重复有效修复所需的基因。该筛选发现了两个新的重组基因，SLX 4和SAW 1，其在从重组中间体去除3'瓣中起作用。3'瓣的去除还取决于结构特异性核酸内切酶复合物Rad 1/Rad 10，并决定细胞对癌症化疗剂的耐受性、基因靶向、端粒完整性、氧化损伤的修复和衰老的抑制。为了解释易错重组的生物化学和分子基础，我们计划定义Saw 1在重组和重组相关生物过程中的生物化学性质，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' flap DNA重建3' flap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密切相关的诱变修复，并为改善癌症治疗铺平道路。

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.