Control of commitment steps in mammalian homologous recombination

描述（由申请人提供）：了解癌细胞的高突变频率是找到预防或延迟疾病发作和随后的化疗耐药方法的关键。DNA双链断裂（dsb）的修复错误可能导致突变，因此了解修复途径如何起作用对于开发减少肿瘤发生频率和化疗耐药出现的新策略至关重要。哺乳动物DSB修复是通过非同源末端连接（NHEJ）和同源重组（HR）介导的。虽然HR使用同源模板进行修复，但NHEJ通过连接dsb起作用。因此，这些途径会产生不同的修复结果，并且细胞中NHEJ和HR的相对使用率是通过一些鲜为人知的过程来调节的。细胞周期似乎是HR通路使用的主要决定因素，HR在G1体细胞中很少使用。通路选择的第二个调节因子是DNA损伤反应因子53BP1。53BP1限制了dsb的切除，这是HR的关键步骤。53BP1因此以牺牲HR为代价促进NHEJ。利用小鼠原代细胞的遗传方法，我们提出测量53BP1如何与包括BRCA1、BLM和Exo1在内的细胞活性相互作用，介导DSB切除，这是调节HR与NHEJ使用的关键步骤。我们将进一步测试是否需要53BP1的限制性切除作用来抑制非分裂细胞中DNA修复缺陷引起的基因组不稳定性和肿瘤发生。最后，我们建议确定BRCA1如何与PALB2相互作用，PALB2是一种帮助招募BRCA2断裂位点的修复因子。BRCA1和PALB2通过预测的线圈区相互作用形成复合体，我们将利用生物物理和细胞生物学技术确定和表征相互作用的确切分子机制。总之，这些研究将促进我们对原代哺乳动物细胞中HR的关键承诺步骤的理解，并为设计合理的治疗策略提供信息，以治疗关键DNA修复基因突变的个体。

DESCRIPTION (provided by applicant): Understanding the high mutation frequency of cancer cells is key to finding ways to prevent or delay the onset of disease and subsequent chemoresistance. Errors in repair of DNA double-strand breaks (DSBs) can cause mutations, hence understanding how repair pathways act is of essential importance for the development of new strategies to reduce the frequency of tumors and the appearance of chemoresistance. Mammalian DSB repair is mediated by nonhomologous end-joining (NHEJ) and homologous recombination (HR). Whereas HR uses a homologous template for repair, NHEJ acts by ligation of DSBs. The pathways therefore produce different repair outcomes, and the relative rate of usage of NHEJ and HR is regulated in cells by processes which are poorly understood. Cell cycle appears to be a major determinant of usage of the HR pathway, with HR used infrequently in G1 somatic cells. A second regulator of pathway choice is the DNA damage response factor, 53BP1. 53BP1 limits resection of DSBs, a key step in HR. 53BP1 therefore promotes NHEJ at the expense of HR. Using genetic approaches in primary mouse cells, we propose to measure how 53BP1 interacts with cellular activities including BRCA1, BLM and Exo1 to mediate DSB resection, a key step regulating the use of HR versus NHEJ. We will furthermore test whether the effect of 53BP1 in limiting resection is required for suppressing genomic instability and tumorigenesis arising from defects in DNA repair in non-dividing cells. Finally, we propose to determine how BRCA1 interacts with PALB2, a repair factor that helps recruit BRCA2 to break sites. BRCA1 and PALB2 form a complex through interactions of predicted coiled-coil regions, and we will identify and characterize the exact molecular mechanism of interaction using biophysical and cell biology techniques. Together, these studies will advance our understanding of critical commitment steps to HR in primary mammalian cells, and inform strategies for the design of rational therapies for treatment of individuals with mutations in key DNA repair genes.