DNA processing roles in DNA Double-strand Break repair by Fanconi Anemia sub-complex AG20

项目摘要 Fanconi贫血(FA)途径形成了一个复杂的DNA修复网络，可以介导肿瘤 癌症患者的侵袭性和治疗抵抗力。尽管已有研究表明FA蛋白 为了支持癌症生存，FA途径生化活性的糟糕表征导致了这一点 表型使得这些蛋白的治疗靶点非常具有挑战性。论小说的人物塑造 FA组分执行的酶作用可以作为有价值的线索，以了解特定的FA蛋白质是如何 能够促进癌症放射治疗的耐药性，并将提供可识别的靶点敏化 将癌细胞转移到传统疗法。具体地说，FA蛋白FANCA、FANCG和FAAP20形成一种 亚复合体(AG20)和FANCA相互作用蛋白BRCA1可能为不稳定的癌症提供关键护理 FANCA固有的DNA/RNA结合和新发现的链退火/交换 活动。这些活动可能有助于DNA双链断裂修复，这能够赋予 放射治疗对癌细胞的生存优势。这项提案的目标是进一步确定 BRCA1和AG20亚复合体的额外DNA和RNA处理活性，以及这些活性是如何 在细胞环境中被用来保存基因组。本项目的目标是：1)鉴定生化 与核酸加工事件相关的BRCA1和AG20亚复合体的活动：定义 AG20和FANCA/BRCA1能够使用DNA/RNA底物进行的活动将可能使用 2)染色质的测定 促进FANCA/BRCA1和AG20介导的修复：FANCA在DSB中的细胞作用 通过对U2OS-DIVA细胞的芯片序列分析，可以对不同的染色质背景进行详细的分析 目前在我们的实验室，并活细胞成像的FANCA募集到特定部位的DNA损伤。通过完成 这些目标，我们期望能够勾画出一个参与修复因子BRCA1的工作模式 和AG20在支持癌症生存和治疗耐药的DNA损伤修复亚群中。

Project Summary The Fanconi Anemia (FA) pathway forms a complex DNA repair network that can mediate tumor aggressiveness and therapy-resistance when dysregulated in cancers. Although FA proteins have been shown to support cancer survival, the poor characterization of FA pathway biochemical activities that contribute to this phenotype has made therapeutic targeting of these proteins very challenging. Characterization of novel enzymatic roles carried out by FA components could serve as valuable clues into how certain FA proteins are able to promote cancer radiotherapy resistance and would provide identifiable targets for sensitization of cancer cells to traditional treatments. Specifically, FA proteins FANCA, FANCG, and FAAP20, which form a sub-complex (AG20), and FANCA-interacting protein BRCA1 may provide critical care of the unstable cancer genome through FANCA’s intrinsic DNA/RNA binding and newly discovered strand annealing/exchange activities. These activities could contribute to DNA double-strand break repair, which is capable of conferring survival advantages to radiotherapy-treated cancer cells. The goal of this proposal is to further determine additional DNA and RNA processing activities of BRCA1 and the AG20 sub-complex, and how these activities are utilized in a cellular context to preserve the genome. The aims of this project are: 1) identify biochemical activities of BRCA1 and the AG20 sub-complex related to nucleic acid processing events: defining the range of activities AG20 and FANCA/BRCA1 are able to perform using DNA/RNA substrates will be possible using highly active recombinant protein that our lab is well-equipped to generate; 2) determine the chromatin landscape that facilitates FANCA/BRCA1 and AG20-mediated repair: FANCA’s cellular role in DSBs across different chromatin contexts can be analyzed in fine detail through ChIP-Seq analysis of U2OS-DiVA cells present in our lab, and live cell imaging of FANCA recruitment to site-specific DNA damage. By accomplishing these goals, we expect to be able to delineate a working model for the participation of repair factors BRCA1 and AG20 in subsets of DNA damage repair that supports cancer survival and therapy-resistance.