INHIBITORS OF COMPENSATORY NHEJ PATHWAYS

描述（由申请人提供）：DNA双链断裂（dsb）是最危险的DNA损伤形式，因为如果修复不当，它们可以形成染色体易位或缺失，如果它们持续未修复，则会导致细胞死亡。DNA DSB修复途径涉及大量蛋白质的协调，其中许多蛋白质对这些途径至关重要。然而，许多DNA修复蛋白可以灭活而不引起明显的DSB修复缺陷。我们认为，在许多情况下，这是由于代偿途径的存在。最显著的例子之一是组蛋白变体H2AX和非同源末端连接（NHEJ）蛋白XLF的联合缺陷。H2AX招募了许多DNA损伤反应蛋白到DSB位点，但缺乏H2AX不会导致NHEJ的明显缺陷。同样，缺乏XLF会导致NHEJ中相对较小的缺陷。然而，H2AX和XLF的联合缺乏导致nhej介导的DSB修复的严重缺陷。这些发现表明H2AX和XLF功能在DNA DSB修复过程中具有代偿或协同功能。我们推断，抑制XLF或与XLF通路相同的蛋白的化合物会在h2ax -缺陷细胞中特异性地抑制NHEJ。在这里，我们将使用一种新的基于细胞系的方法来检测DNA DSB修复，以开发一种高通量筛选可以抑制NHEJ的化合物。在与NIH化学基因组学中心的合作中，我们将使用该筛选来识别在h2ax缺陷细胞中特异性抑制NHEJ的化学物质。我们还将制定确定目标的方法

DESCRIPTION (provided by applicant): DNA double strand breaks (DSBs) are the most dangerous forms of DNA damage, as they can form chromosomal translocations or deletions if improperly repaired, or cause cell death if they persist unrepaired. DNA DSB repair pathways involve coordination of a large cohort of proteins, many of which are essential for these pathways. However, many DNA repair proteins can be inactivated without causing significant DSB repair defects. We reason that in many cases this is due to the presence of compensatory pathways. One of the most dramatic examples is revealed by the combined deficiency in the histone variant H2AX and the non-homologous end joining (NHEJ) protein XLF. H2AX recruits many DNA damage response proteins to DSB sites, but deficiency in H2AX does not cause an overt defect in NHEJ. Similarly, deficiency in XLF results in relatively minor defects in NHEJ. However, combined deficiency of H2AX and XLF results in a severe defect in NHEJ-mediated DSB repair. These findings suggest that H2AX and XLF function have compensatory or synergistic functions during DNA DSB repair. We reason that compounds that inhibit XLF or proteins in the same pathway as XLF would inhibit NHEJ specifically in H2AX-deficient cells. Here, we will use a novel cell line based approach for assaying DNA DSB repair to develop a high throughput screen for compounds that can inhibit NHEJ. In collaboration with the NIH Chemical Genomics Center, we will use this screen to identify chemicals that inhibit NHEJ specifically in H2AX-deficient cells. We will also establish approaches for identifying the targets of these compounds. Completion of these studies will be important in several ways. First, our findings will establish that agents that do not affect DSB repair in normal cells may affect DSB repair and genomic stability in rare cells that have lesions that lead to reduced H2AX expression. Second, as many tumors inactivate H2AX, compounds identified through our screen could be used therapeutically to sensitize these tumor cells to genotoxic agents. Finally, in addition to H2AX, there are other DNA repair proteins whose inactivation does not lead to severe DSB repair defects. Our approach could be used to both establish the existence and identify the components of compensatory pathways for these proteins, and to identify compounds that inhibit these pathways.