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

DNA 处理在范可尼贫血亚复合物 AG20 DNA 双链断裂修复中的作用

• 批准号: 10444900
• 负责人: Anna Louise Palovcak
• 金额: $ 4.53万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10444900
• 关键词: Address; Affinity; Appearance; BRCA1 Protein; BRCA1 gene; Binding; Biochemical; Biochemical Pathway; Biological Assay; Catalysis; Catalytic Domain; Cell physiology; Cells; ChIP-seq; Chromatin; Complex; Confocal Microscopy; Critical Care; DNA; DNA Damage; DNA Double Strand Break; DNA Interstrand Crosslinking; DNA Repair; DNA analysis; Data; Double Strand Break Repair; Event; Excision; Fanconi Anemia Complementation Group A Protein; Fanconi Anemia Complementation Group G Protein; Fanconi Anemia pathway; Fanconi anemia protein; Fanconi' s Anemia; G2 Phase; Genetic Recombination; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Human Biology; Hybrids; In Vitro; Lesion; Maintenance; Malignant Neoplasms; Mediating; Mission; Modeling; Molecular; National Heart, Lung, and Blood Institute; Nucleic Acids; Paint; Pathway interactions; Phenotype; Physiological; Proteins; Public Health; RNA; RNA Binding; RNA Processing; Radiation therapy; Reaction; Recombinant Proteins; Research; Resistance; Role; Site; Structure; System; Testing; Work; base; cancer cell; cancer genome; cancer radiation therapy; cancer survival; cancer therapy; cancer type; homologous recombination; human disease; live cell imaging; member; novel; nuclease; preservation; reconstitution; recruit; refractory cancer; repair function; repaired; targeted treatment; therapeutic target; therapy resistant; traditional therapy; tumor

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.

项目摘要 范可尼贫血（FA）通路形成了一个复杂的DNA修复网络，可以介导肿瘤 癌症调节失调时的攻击性和治疗抵抗性。虽然FA蛋白已被证明 为了支持癌症存活，FA途径生化活性的不良表征有助于这一点， 表型使得这些蛋白质的治疗靶向非常具有挑战性。小说人物塑造 由FA组分进行的酶促作用可以作为了解某些FA蛋白是如何被激活的有价值的线索。 能够促进癌症放射治疗抗性，并将提供可识别的靶点， 癌细胞到传统治疗。具体地，FA蛋白FANCA、FANCG和FAAP 20，它们形成一个由FA蛋白FANCA、FANCG和FAAP 20组成的结构。 亚复合物（AG 20）和FANCA相互作用蛋白BRCA 1可以提供不稳定癌症的重症监护 通过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，这是能够赋予 放射治疗治疗的癌细胞的生存优势。该提案的目标是进一步确定 BRCA 1和AG 20亚复合物的额外DNA和RNA加工活性，以及这些活性如何 在细胞环境中用于保存基因组。本项目的目标是：1）鉴定生物化学 与核酸加工事件相关的BRCA 1和AG 20亚复合物的活性： AG 20和FANCA/BRCA 1能够使用DNA/RNA底物进行的活动将可能使用 我们实验室有能力生产的高活性重组蛋白; 2）确定染色质 促进FANCA/BRCA 1和AG 20介导的修复的景观：FANCA在DSB中的细胞作用 通过U2 OS-DiVA细胞的ChIP-Seq分析， 目前在我们的实验室，和活细胞成像的FANCA招募位点特异性DNA损伤。通过完成 这些目标，我们希望能够描绘一个工作模型的参与修复因子BRCA 1 和AG 20在支持癌症存活和治疗抗性的DNA损伤修复亚组中的作用。