Homologous recombination mechanisms in mammalian cells

 描述（由申请人提供）：我的实验室对哺乳动物细胞中同源重组（HR）的机制和因素有着长期的兴趣。我们确定HR是哺乳动物细胞中DNA双链断裂（DSB）的主要修复机制，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，小鼠SPO 11蛋白引入DSB来启动减数分裂HR，并且在小鼠减数分裂HR期间发生非孟德尔信息传递（基因转换）。HR在体细胞中的重要性特别强调了HR基因突变与人类疾病，特别是癌症的关联，而在生殖细胞中，HR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的发现基础上，长期目标是了解有丝分裂和减数分裂细胞中的HR机制和因子。 我们以前的观察表明，减数分裂基因转换发生更广泛地通过减数分裂HR热点比预期的相对集中的DSB形成，这对热点的进化寿命的影响。我们将探讨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，以及序列差异如何影响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至关重要的，但如果它导致杂合性丧失，则它在有丝分裂细胞中可能是有害的。然而，有丝分裂同源重组的研究已经落后于那些在减数分裂细胞。我们将确定减数分裂和有丝分裂同源HR的机制如何比较，以及不同的细胞环境如何影响DNA交易。 在减数分裂过程中，DSB的数量和位置需要严格调节。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，包括性染色体共享的假常染色体区域。在减数分裂中，HR对DSB进行适当的修复是至关重要的，因为通常每个同源物上会发生一到两次交换，而大多数剩余的修复被认为会产生同源物间的非交换。我们将讨论ATM的丢失是否会导致异常的DSB修复结果。 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。我们已经开始探索额外的假定HR因素的功能，并计划一个综合的方法来了解这些因素在动物中的作用与已知的HR因素。最后，选择DSB进行HR或更多的诱变性非同源修复似乎是在最初的末端处理步骤，称为末端切除控制。然而，这一过程及其控制在哺乳动物细胞中知之甚少。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.