Homologous recombination mechanisms in mammalian cells

哺乳动物细胞中的同源重组机制

• 批准号: 9923699
• 负责人: Maria Jasin
• 金额: $ 55.69万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-03 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9923699
• 关键词: Address; Affect; Animals; Cells; Chromosomes; DNA; DNA Double Strand Break; DNA Repair; DNA lesion; Defect; Development; Double Strand Break Repair; Embryo; Excision; Fertility; Frequencies; Gene Conversion; Genes; Genetic Recombination; Genome; Genomics; Germ Cells; Goals; Homologous Gene; Human; Lead; Lesion; Longevity; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Mammals; Mammary Neoplasms; Meiosis; Mismatch Repair; Mitotic; Mus; Mutation; Nucleotides; Outcome; Pattern; Play; Positioning Attribute; Process; Pseudoautosomal Region; Regulation; Resolution; Role; Sex Chromosomes; Somatic Cell; Spo11 protein; Spontaneous abortion; Sterility; Transact; Tumor Suppressor Proteins; ataxia telangiectasia mutated protein; homologous recombination; human disease; insight; interest; malignant breast neoplasm; ovarian neoplasm; public health relevance; repaired; segregation; tumor; tumorigenesis

 DESCRIPTION (provided by applicant): My lab has a long-standing interest in mechanisms and factors involved in homologous recombination (HR) in mammalian cells. We established that HR is a major repair mechanism in mammalian cells for DNA double-strand breaks (DSBs) and that breast and ovarian tumor suppressors are HR factors. In addition, we demonstrated with my colleague Scott Keeney that the mouse SPO11 protein introduces DSBs to initiate meiotic HR and that the non-Mendelian transfer of information - gene conversion - occurs during meiotic HR in the mouse. The importance of HR in somatic cells is particularly emphasized by the association of mutations in HR genes with human disease, especially cancer, while in germ cells, mistakes in HR can lead to miscarriage and developmental defects. The application builds on our earlier discoveries with the long-term goals of understanding HR mechanisms and factors in mitotic and meiotic cells. Our previous observations demonstrated that meiotic gene conversion occurs more broadly through meiotic HR hotspots than expected from relatively focused DSB formation, which has implications for the evolutionary longevity of hotspots. We will explore possible mechanisms that could lead to this pattern of gene conversion, focusing on the role of mismatch repair factors, as well as how sequence divergence affects HR frequency and outcomes. While HR between homologous chromosomes is critical in meiotic cells, it can be deleterious in mitotic cells if it leads to loss of heterozgosity. However, studies of mitotic interhomolog recombination have lagged behind those in meiotic cells. We will determine how mechanisms of meiotic and mitotic interhomolog HR compare and how the different cellular contexts impact DNA transactions. During meiosis, DSB numbers as well as position need to be tightly regulated. The ATM kinase plays a critical role in regulating meiotic DSB numbers; we will address whether ATM also controls the spatial regulation of DSB formation, including on the pseudoautosomal region shared by the sex chromosomes. Proper DSB repair by HR is critical in meiosis, as typically one to two crossovers occur on each homolog, with much of the remaining repair presumed to give rise to interhomolog noncrossovers. We will address whether loss of ATM leads to aberrant DSB repair outcomes. Many HR factors are crucial in mammals as their loss can lead to embryonic lethality, developmental defects, sterility, or tumorigenesis. We have begun to explore the function of additional presumptive HR factors and plan an integrated approach to understand the role of these factors in the animal in relation to that of known HR factors. Finally, the choice for a DSB to undergo HR or more mutagenic nonhomologous repair appears to be controlled at the initial end processing step, termed end resection. However, this process and its control are poorly understood in mammalian cells. We will develop nucleotide resolution end resection analysis to gain insight into this key control step.

 描述（由适用提供）：我的实验室对哺乳动物细胞中同源重组（HR）涉及的机制和因素具有长期的兴趣。我们确定HR是DNA双链断裂（DSB）的哺乳动物细胞中的主要修复机制，并且乳腺和卵巢肿瘤补充剂是HR因素。此外，我们与我的同事Scott Keeney一起证明了小鼠SPO11蛋白引入DSB以启动减数分裂HR，并且在小鼠中减数分裂HR期间发生了信息的非孟德尔信息转移 - 基因转换 - 发生。人力资源基因与人类疾病（尤其是癌症）的突变相关性特别强调了人力资源在体细胞中的重要性，而在生殖细胞中，人力资源中的错误可能导致流产和发育缺陷。该应用程序建立在我们早期发现的基础上，其长期目标是理解有丝分裂和减数分裂细胞中的人力资源机制和因素。我们以前的观察结果表明，减数分裂基因的转化是通过减数分裂的HR热点比相对集中的DSB形成所预期的更广泛的，这对热点的进化寿命具有影响。我们将探索可能导致这种基因转化模式的可能机制，重点关注不匹配修复因子的作用，以及序列差异如何影响HR频率和结果。尽管同源染色体之间的HR在减数分裂细胞中至关重要，但如果它导致杂化性丧失，则可以在有丝分裂细胞中删除。但是，有丝分裂的研究研究落后于减数分裂细胞的研究。我们将确定减数分裂和有丝分裂的机理如何比较以及不同的细胞环境如何影响DNA交易。在减数分裂期间，需要严格调节DSB的数字和位置。 ATM激酶在控制减数分裂DSB数中起着至关重要的作用。我们将解决ATM是否还控制DSB形成的空间调节，包括性别染色体共享的假阳性次素图区域。人力资源的正确DSB修复对减数分裂至关重要，因为通常在每个同源物上发生一到两个交叉，其余的修复假定会引起本体间非交叉。我们将解决ATM损失是否导致DSB修复结果异常。许多人力资源因素在哺乳动物中至关重要，因为它们的损失会导致胚胎致死性，发育缺陷，无菌性或肿瘤发生。我们已经开始探索其他假定人力资源因素的功能，并计划了一种综合方法，以了解这些因素在动物中与已知HR因子相关的作用。最后，DSB进行HR或更多诱变非脑清单修复的选择似乎是在初始末端处理步骤中控制的，称为终端切除。但是，在哺乳动物细胞中，该过程及其控制知之甚少。我们将开发核苷酸分辨率末端切除分析，以深入了解这一关键控制步骤。