Control of commitment steps in mammalian homologous recombination

哺乳动物同源重组中承诺步骤的控制

• 批准号: 9762020
• 负责人: Samuel Bunting
• 金额: $ 34.1万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762020
• 关键词: Affect; Appearance; BRCA1 gene; BRCA2 gene; Biophysics; Bone Marrow; Brain; Cancer Cell Growth; Cell Cycle; Cell physiology; Cells; Cellular biology; Coiled-Coil Domain; Complex; Cytotoxic Chemotherapy; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA Sequence Alteration; Databases; Defect; Development; Double Strand Break Repair; EXO1 gene; Embryo; Environment; Excision; Frequencies; G0 Phase; G1 Phase; Genes; Genetic; Genome; Genomic Instability; Goals; Hematopoietic; Hematopoietic stem cells; Higher Order Chromatin Structure; Homodimerization; Incidence; Individual; Interphase Cell; Learning; Ligation; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Methods; Mitotic; Modeling; Molecular; Mus; Mutation; Neurons; Nonhomologous DNA End Joining; Normal Cell; Onset of illness; Operating System; Outcome; Pathologic; Pathway interactions; Patients; Physiological; Population; Reagent; Repression; Resources; Role; Site; Site-Directed Mutagenesis; Somatic Cell; Structural Models; Structure; System; Techniques; Testing; Transgenic Organisms; Work; X-Ray Crystallography; base; cancer cell; cancer therapy; design; experimental study; genetic approach; genome integrity; homologous recombination; in vivo; p53-binding protein 1; prevent; public health relevance; reconstitution; recruit; relating to nervous system; repaired; response; success; therapy design; three dimensional structure; tumor; tumorigenesis; variant of unknown significance

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.

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性的方法的关键。DNA双链断裂（DSB）修复中的错误可能导致突变，因此了解修复途径如何起作用对于开发新策略以降低肿瘤发生率和化学耐药性的出现至关重要。哺乳动物DSB修复是由非同源末端连接（NHEJ）和同源重组（HR）介导的。而HR使用同源模板进行修复，NHEJ通过DSB的连接起作用。因此，这些途径产生不同的修复结果，并且NHEJ和HR的相对使用率在细胞中通过知之甚少的过程进行调节。细胞周期似乎是使用HR途径的主要决定因素，HR在G1体细胞中很少使用。第二个调节途径选择的是DNA损伤反应因子，53BP1。53BP1限制切除DSB，在HR.53BP1的关键步骤，因此促进NHEJ在HR.Using在原代小鼠细胞中的遗传方法，我们建议测量53BP1如何与细胞活动，包括BRCA1，BLM和Exo1介导DSB切除，调节HR与NHEJ的使用的关键步骤。我们将进一步测试53BP1在限制切除中的作用是否是抑制基因组不稳定性和由非分裂细胞中DNA修复缺陷引起的肿瘤发生所必需的。最后，我们建议确定BRCA1如何与PALB 2相互作用，PALB 2是一种修复因子，有助于招募BRCA2来破坏网站。BRCA1和PALB2通过预测的卷曲螺旋区域的相互作用形成复合物，我们将使用生物物理学和细胞生物学技术鉴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关键承诺步骤的理解，并为设计合理治疗策略提供信息，用于治疗关键DNA修复基因突变的个体。