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中染色体断裂同源修复期间影响遗传丢失程度的因素和途径。为此，我们正在研究由罕见的切割核酸内切酶l-Scel产生的断裂的修复。我们最近发现，在一些基因的缺陷导致相对更多的遗传损失在同源修复这样的断裂。我们推测，这种影响的遗传损失可能是由于中断的同源修复的离散步骤的控制。一个这样的步骤可以是5'至3' DNA末端切除，这是因为通过该切除产生的单链尾具有促进更多诱变修复途径的潜力，并且因为单链尾似乎对于DNA损伤诱导的细胞周期检查点是重要的。基因转换期间的复制是另一个重要的机械步骤，可以通过确定基因转换期间转移的遗传信息量来影响修复期间遗传丢失的程度。我们建议测试的假设，5'到3'切除和/或复制步骤的同源修复的机械控制是至关重要的限制染色体断裂修复过程中的遗传损失。具体目标是：1.为了验证限制5'至3'切除和/或复制对于抑制基因转换期间的遗传丢失很重要的假设。为此，我们将开发和分析一系列重组报告基因，这些报告基因在修复事件所需的切除程度与复制程度上不同。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RAD 51、BRCA 1和BRCA 2的细胞中分析目标1中描述的报告基因。3.在哺乳动物细胞的多种遗传背景下，以物理方式确定染色体断裂的5'至3'切除的频率和程度。这些物理实验对于理解切除的控制如何影响遗传损失至关重要。与公共卫生的相关性：我们的目标是了解受损的DNA是如何修复的，因为这个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遗传丢失的过程以及可能增加利用DNA损伤剂的癌症治疗功效的药物的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.