Mechanistic steps of homologous repair in mammalian cells

描述(申请人提供)：这项研究的长期目标是了解在哺乳动物细胞染色体断裂的同源修复过程中影响遗传丢失程度的因素和途径。为此，我们正在研究修复由罕见的切割内切酶L-SCEL产生的断裂。我们最近发现，在这些断裂的同源修复过程中，许多基因的缺陷会导致相对更多的基因丢失。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的控制中断所致。其中一个步骤可能是5‘到3’DNA末端切除，这既是因为这种切除产生的单链尾巴有可能促进更多的突变修复途径，也因为单链尾巴似乎对DNA损伤诱导的细胞周期检查点很重要。基因转化过程中的复制是另一个重要的机械性步骤，它可以通过确定基因转化过程中转移的遗传信息量来影响修复过程中遗传损失的程度。我们建议检验这一假设，即同源修复的5‘到3’切除和/或复制步骤的机械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。其具体目的是：1.验证限制5‘至3’切除和/或复制对于抑制基因转化过程中的遗传损失是重要的假设。为此，我们将开发和分析一系列重组记者，这些重组记者的切除程度与修复事件所需的复制程度不同。2.检验个体遗传因素可能通过影响切除和/或复制的控制来影响遗传丢失的假设。为此，我们建议在缺乏RAD51、BRCA1和BRCA2的细胞中分析目标1中描述的报告。3.从物理上确定哺乳动物细胞在不同遗传背景下5‘至3’切除染色体断裂的频率和程度。这些物理实验对于理解切除的控制如何影响基因丢失是基本的。与公众健康相关：我们的目标是了解受损的DNA是如何修复的，因为这一过程中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的基因丢失过程以及对可能提高利用DNA损伤剂进行癌症治疗的疗效的药物潜在靶点的机械表征是重要的。

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.