Homologous recombination mechanisms in mammalian cells

描述(申请人提供)：我的实验室长期以来一直对哺乳动物细胞中同源重组(HR)的机制和因素感兴趣。我们证实，HR是哺乳动物细胞DNA双链断裂(DSB)的主要修复机制，而乳腺和卵巢肿瘤抑制因子是HR因子。此外，我们与我的同事Scott Keeney一起证明了小鼠SPO11蛋白引入DSB来启动减数分裂HR，并且信息的非孟德尔转移-基因转换-发生在小鼠的HR减数分裂过程中。在体细胞中，HR基因的突变与人类疾病，特别是癌症的关联特别强调了HR的重要性，而在生殖细胞中，HR的错误可能导致流产和发育缺陷。这项应用建立在我们早期发现的基础上，长期目标是了解有丝分裂和减数分裂细胞中的HR机制和因素。我们之前的观察表明，减数分裂基因转换通过减数分裂HR热点发生的范围比预期的相对集中的DSB形成更广泛，这对热点的进化寿命有一定的意义。我们将探索可能导致这种基因转换模式的可能机制，重点是错配修复因子的作用，以及序列分歧如何影响HR频率和结果。虽然同源染色体之间的HR在减数分裂细胞中是关键的，但如果它导致异质性的丧失，在有丝分裂细胞中可能是有害的。然而，有丝分裂同系物重组的研究已经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同系物之间的机制是如何比较的，以及不同的细胞环境如何影响DNA交易。在减数分裂过程中，DSB的数量和位置需要严格控制。ATM激酶在调节减数分裂DSB数量中起着关键作用；我们将讨论ATM是否也控制DSB形成的空间调节，包括在性染色体共享的伪常染色体区域。HR正确的DSB修复在减数分裂中是至关重要的，因为通常在每个同源上发生一到两个交换，其余的修复被认为会导致同源间的非交换。我们将解决ATM丢失是否会导致DSB修复结果异常的问题。许多HR因子在哺乳动物中是至关重要的，因为它们的缺失可能导致胚胎死亡、发育缺陷、不育或肿瘤发生。我们已经开始探索其他假定的HR因子的功能，并计划一种综合的方法来了解这些因子在动物中的作用与已知HR因子的作用。最后，DSB接受HR或更多诱变的非同源修复的选择似乎在最初的末端处理步骤，称为末端切除时受到控制。然而，在哺乳动物细胞中对这一过程及其控制知之甚少。我们将开发核苷酸解析末端切除分析，以深入了解这一关键控制步骤。

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.