Mechanisms of Error Prone Repair of DNA Breaks

描述(申请人提供)：DNA双链断裂S是由多种遗传毒性物质引起的，包括电离辐射和用于治疗癌症的化学物质。消除DSB是通过独特的无错误和容易出错的途径进行的。同源重组的修复在很大程度上是无错误的，并由RAD52上位组基因介导。需要Ku异源二聚体的非同源末端连接(NHEJ)可以有效地重新连接断裂，但偶尔会丢失或获得DNA信息。新出现的证据揭示了一种新的DNA末端连接机制，该机制独立于Rad52和Ku蛋白。这一新的DSB修复途径通过微同源介导的DNA单链碱基配对封闭DNA断裂，随后是DNA瓣的核溶解修剪、DNA缺口填充和DNA连接，产生的产物几乎总是与DNA缺失有关。这种高度容易出错的DSB修复途径被称为微同源介导的末端连接(MMEJ)。分析MMEJ的机制是非常有兴趣的，因为它可能通过基因缺失来破坏基因组的稳定。我们已经开发了一种基于酵母菌的生物系统来创造特定的双链断裂，这些断裂可以优先由MMEJ修复。这种MMEJ分析的可用性为分析这种反应的遗传要求提供了一个独特的机会。事实上，这个生物系统已经让我们能够识别出几种影响MMEJ效率的基因产物。这项建议的重点是描绘这些基因产物在MMEJ中的功能，并确定影响这一过程的其他基因。由于DSB修复的成分从酵母到人类都是保守的，我们的研究获得的见解将对剖析人类细胞中的相同过程有价值，并将与公共卫生相关。

DESCRIPTION (provided by applicant): DNA double-strand breaks (DSB)s are induced by a variety of genotoxic agents, including ionizing radiation and chemicals used for treating cancers. The elimination of DSBs proceeds via distinctive error-free and error-prone pathways. Repair by homologous recombination is largely error-free and mediated by the RAD52 epistasis group genes. Non-homologous end joining (NHEJ) that requires the Ku heterodimer can efficiently rejoin breaks, with occasional loss or gain of DNA information. Emerging evidence has unveiled a novel DNA end-joining mechanism that is independent of Rad52 and Ku proteins. This novel pathway of DSB repair seals DNA breaks by microhomology-mediated base-pairing of DNA single strands, followed by nucleolytic trimming of DNA flaps, DNA gap filling, and DNA ligation, yielding products that are almost always associated with DNA deletion. This highly error-prone DSB repair pathway is termed microhomology-mediated end joining (MMEJ). Dissecting the mechanism of MMEJ is of great interest because of its potential to destabilize the genome through gene deletions. We have developed a Saccharomyces-based biological system to create specific double-strand breaks that are preferentially repaired by MMEJ. The availability of this MMEJ assay presents a unique opportunity to dissect the genetic requirement of this reaction. In fact, this biological system has already allowed us to identify several gene products that affect the efficiency of MMEJ. The focus of this proposal is to delineate the functions of these gene products in MMEJ and to identify additional genes that influence this process. Since the components of DSB repair are conserved from yeast to humans, the insights garnered from our research will be valuable for dissecting the equivalent process in human cells and will be of relevance to public health.