Defining the mechanism of Shieldin complex in DNA end joining

定义 Shieldin 复合物在 DNA 末端连接中的机制

• 批准号: 10066830
• 负责人: Joshua Heyza
• 金额: $ 6.49万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-12 至 2023-08-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10066830
• 关键词: Address; Affinity; BRCA1 gene; BRCA2 gene; Binding; Biological; Biological Assay; CRISPR therapeutics; CRISPR/Cas technology; Cell Cycle; Cell Line; Cell physiology; Cells; Chemicals; Clinical; Clinical Research; Complex; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; Dangerousness; Defect; Development; Disease; Double Strand Break Repair; Event; Excision; Foundations; Future; G2 Phase; Genes; Genetic; Genome Stability; Human; Immune; Immune System Diseases; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; In Vitro; Lasers; Lead; Length; Lesion; Malignant Neoplasms; Maps; Mechanics; Mediating; Mediator of activation protein; Meiosis; Modeling; Molecular; Mutation; Nonhomologous DNA End Joining; Outcomes Research; Pathway interactions; Physiological Processes; Poly(ADP-ribose) Polymerases; Proteins; Resistance; Resolution; Role; S Phase; Signal Transduction; Sister Chromatid; Syndrome; Work; cancer cell; genome editing; genome integrity; homologous recombination; human disease; imaging approach; improved; in vivo; inhibitor/antagonist; insight; live cell imaging; molecular imaging; novel anticancer drug; novel therapeutic intervention; p53-binding protein 1; preservation; protein protein interaction; recruit; repaired; response; single molecule; tumor

Project Summary: Human cells regularly acquire DNA double strand breaks (DSB) due to exogenous and endogenous chemicals and as part of natural physiological processes (e.g. immune class switch recombination and meiotic crossover events). Therefore, cells have evolved highly regulated DSB repair pathways which serve to protect from particularly dangerous threats to genome integrity. It is for this reason that mutations in pathways responsible for proper DSB recognition, processing, and resolution contribute to a variety of human diseases including immune disorders, a variety of genetic syndromes, and cancer development. In general, cells have access to two major pathways for DSB resolution, homologous recombination (HR) and non-homologous end joining (NHEJ). A key regulator of DSB repair pathway choice is the 53BP1 protein which promotes NHEJ by blocking extensive resection at a DSB which would favor HR. The Shieldin complex (composed of SHLD1, SHLD2, SHLD3 & REV7) was recently identified as a downstream effector of 53BP1 signaling. Shieldin effects 53BP1’s anti-HR functions by binding ssDNA overhangs at DSBs and blocking end resection. These observations lead to the following conundrum: how can NHEJ, which prefers short overhangs or blunt DNA ends, be promoted by a ssDNA binding complex? I propose to define the molecular mechanism underlying Shieldin function in DSB repair using single-molecule (SM) imaging approaches. To this end, I will generate a panel of HaloTagged DNA damage response (DDR) proteins using CRISPR/Cas9 mediated genome editing that will be used for SM live- cell imaging. With these tagged proteins, I will dissect the regulatory mechanisms that control Shieldin recruitment to DNA DSBs in vivo and define the molecular determinants for Shieldin binding at DSBs in vitro. Generating a quantitative model of Shieldin function in regulating DSB repair will reveal the fundamental mechanics of how Shieldin controls DSB repair pathway choice. As such, results from these studies will have the potential for significant impact in the field as Shieldin’s role in DSB repair is incompletely understood. Additionally, generating a quantitative model of Shieldin function could lead to the uncovering of novel therapeutic approaches to target Shieldin function for clinical benefit.

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