Mechanistic steps of homologous repair in mammalian cells

描述（由申请人提供）：本研究的长期目标是了解哺乳动物细胞染色体断裂同源修复过程中影响遗传损失程度的因素和途径。为此，我们正在研究由罕见的切割内切酶l-Scel产生的断裂的修复。我们最近表明，在同源修复这种断裂时，许多基因的缺陷导致相对更多的遗传损失。我们假设这种对遗传损失的影响可能是由于同源修复的离散步骤控制的中断。其中一个步骤可能是5‘到3’ DNA末端切除，因为这种切除产生的单链尾巴有可能促进更多的诱变修复途径，而且单链尾巴似乎对DNA损伤诱导的细胞周期检查点很重要。基因转换过程中的复制是另一个重要的机制步骤，它可以通过决定基因转换过程中传递的遗传信息的数量来影响修复过程中遗传损失的程度。我们建议验证这一假设，即同源修复的5‘到3’切除和/或复制步骤的机制控制对于限制染色体断裂修复过程中的遗传损失至关重要。具体目标是：1。为了验证限制5‘到3’的切除和/或复制对抑制基因转化过程中的遗传损失很重要的假设。为此，我们将开发和分析一系列重组报告基因，它们在修复事件所需的切除程度和复制程度上有所不同。2. 检验单个遗传因素可能通过影响切除和/或复制的控制来影响遗传损失的假设。为此，我们建议分析Aim 1中描述的RAD51、BRCA1和BRCA2缺失细胞中的报告基因。3. 在哺乳动物细胞的各种遗传环境中，物理确定染色体断裂的5‘至3’切除的频率和程度。这些物理实验是理解控制切除如何影响遗传损失的基础。与公共卫生相关：我们的目标是了解受损DNA是如何修复的，因为这一过程的失败会导致遗传信息的丢失。这一目标对于理解癌症发展过程中基因丢失的过程，以及对潜在靶点的机制表征非常重要，这些靶点可以提高利用DNA损伤剂治疗癌症的疗效。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the factors and pathways that influence the extent of genetic loss during homologous repair of chromosomal breaks in mammalian cells. For this, we are studying the repair of a break generated by the rare cutting endonuclease, l-Scel. We have recently shown that deficiencies in a number of genes result in relatively more genetic loss during homologous repair of such breaks. We hypothesize that such effects on genetic loss could be due to the disruption in the control of discrete steps of homologous repair. One such step could be 5' to 3' DNA end resection, both since single stranded tails generated by this resection have the potential to promote more mutagenic repair pathways, and since single stranded tails appear to be important for DNA damage-induced cell cycle checkpoints. Replication during gene conversion is another important mechanistic step that could influence the extent of genetic loss during repair by determining the amount of genetic information transferred during gene conversion. We propose to test the hypothesis that the mechanistic control of the 5' to 3' resection and/or replication steps of homologous repair are critical for limiting genetic loss during chromosomal break repair. The specific aims are: 1. To test the hypothesis that limiting 5' to 3' resection and/or replication is important to suppress genetic loss during gene conversion. For this, we will develop and analyze a series of recombination reporters, which differ in the degree of resection versus replication required for the repair event. 2. To test the hypothesis that individual genetic factors may influence genetic loss by affecting the control of resection and/or replication. For this, we propose to analyze the reporters described in Aim 1 in cells deficient for RAD51, BRCA1, and BRCA2. 3. To physically determine the frequency and extent of 5' to 3' resection of a chromosomal break in a variety of genetic contexts in mammalian cells. These physical experiments are fundamental to an understanding of how the control of resection may influence genetic loss. Relevance to public health: Our objective is to understand how damaged DNA is repaired, since failures in this process results in loss of genetic information. This objective is important for understanding the process of genetic loss during cancer development, as well as for a mechanistic characterization of potential targets of drugs that could increase the efficacy of cancer treatments that utilize DNA damaging agents.