Mechanism of Meiotic Recombination

减数分裂重组机制

• 批准号: 10702279
• 负责人: MICHAEL J LICHTEN
• 金额: $ 130万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702279
• 关键词: Biochemical; Biological Models; Biological Process; Chromosome Structures; Chromosomes; Complex; Congenital Abnormality; Cruciform DNA; DNA Damage; DNA Double Strand Break; DNA Repair; Development; Double Strand Break Repair; Ensure; Event; Failure; Genetic; Genetic Recombination; Genome; Growth; High-Throughput Nucleotide Sequencing; Homologous Gene; Human; Infertility; Lead; Location; Malignant Neoplasms; Meiosis; Meiotic Recombination; Methods; Mitosis; Mitotic Cell Cycle; Molecular; Molecular Chaperones; Mosaicism; Pathway interactions; Population; Process; Proteins; Recombinants; Regulation; Research; Saccharomyces cerevisiae; Saccharomycetales; Sister Chromatid; Somatic Cell; Testing; Topoisomerase; Work; Yeasts; base; helicase; homologous recombination; insight; member; migration; novel; prevent; recruit; repaired; tool; topoisomerase IIIalpha; transmission process

Meiotic double-strand DNA break (DSB) repair by homologous recombination occurs via multiple processes defined by distinct decisions points. One important decision involves partner choice, between recombining with the sister chromatid (the dominant repair partner during mitosis) or with the homolog (the homologous chromosome of different parental origin, the preferred partner during meiosis). Another important decision involves recombination pathway choice, between producing crossovers, where flanking chromosome sequences are exchanged, or noncrossovers. A signature contribution of our group was the demonstration that crossover and noncrossover recombination proceed via different mechanisms that diverge after initial stages of strand invasion, and that feature different biochemical activities and genetic requirements. Work during previous review periods had shown that the conserved Sgs1-Top3-Rmi1 helicase-topoisomerase complex (STR) is responsible for partitioning early recombination events between noncrossover and crossover pathways. Sgs1-Top3-Rmi1 is the yeast homolog of the mammalian BLM helicase-Top3alpha-BLAP75 complex, implicated in cancer avoidance and recombination control in humans. We showed that all three members of the yeast complex are essential for normal recombination partner choice and for population of regulated meiotic crossover and noncrossover recombination pathways. Based on these findings, we hypothesized that STR, by promoting frequent disassembly of early strand invasion intermediates, acts as a chaperone for early recombination intermediates. We hypothesized that these repeated cycles of strand invasion and disassembly would result in template switching, which in turn would lead to recombinants with mosaic parental strand contributions. This hypothesis has now been confirmed by high-throughput sequencing of recombinants that occur in a highly polymorphic test interval; more than 2/3 of recombinants display clear evidence for template switching, multiple strand invasions, or both. In addition, we uncovered evidence for activities specific to the crossover pathway, including branch migration (2/3 of crossovers) and exonucleolytic gap-formation (1/3 of crossovers). Current work is aimed at determining the proteins responsible for these activities. Other work is aimed at confirming branch migration by mapping the location of Holliday junctions in recombination using a novel method we have developed to specifically purify Holliday junction-containing intermediates. Finally, we are studying how chromosome structure, specifically the meiotic chromosome axis, contributes to the regulation of recombination. A meiosis-specific subset of chromosome axis components, the Hop1 and Red1 proteins, are important for meiotic DSB formation and partner choice, and are enriched in some regions of the genome relative to others. Using a novel method to recruit axis proteins to regions that are normally depleted of these proteins, we have shown that high concentrations of the Hop1 protein are necessary and sufficient for meiotic DSB formation, but the recombination events initiated by these DSBs do not follow canonical meiotic recombination pathways. We are currently determining the mechanism by which Hop1 promotes DSB formation, and what additional factors are needed for Hop1-dependent DSBs to be repaired by canonical meiotic recombination mechanisms.

通过同源重组进行的减数分裂双链DNA断裂（DSB）修复是通过由不同决策点定义的多个过程发生的。一个重要的决定涉及合作伙伴的选择，与姐妹染色单体（有丝分裂期间的主要维修伴侣）或同源物（不同亲本起源的同源染色体（减数分裂过程中的首选伴侣）之间的重新组合）。另一个重要的决策涉及重组途径选择，在产生跨界，侧翼染色体序列或非交叉之间。我们小组的签名贡献是，证明了跨界和非交叉重组通过不同的机制进行的，这些机制在链入侵的初始阶段发生分歧，并且具有不同的生化活动和遗传要求。在先前的审查期间的工作表明，保守的SGS1-TOP3-RMI1解旋酶 - toisomerase复合酶（STR）负责分区非交叉和交叉途径之间的早期重组事件。 SGS1-TOP3-RMI1是哺乳动物BLM解旋酶-TOP3Alpha-Blap75复合物的酵母同源物，与人类的癌症回避和重组控制有关。我们表明，酵母菌络合物的所有三个成员对于正常的重组伴侣选择以及受调节的减数分裂跨界和非交叉重组途径的人群至关重要。基于这些发现，我们假设STR通过促进早期链入侵中间体的频繁拆卸来充当早期重组中间体的伴侣。我们假设这些重复的链入侵和拆卸的循环将导致模板切换，这反过来又导致具有马赛克父母链贡献的重组。现在，通过在高度多态测试间隔中发生的重组分子的高通量测序证实了这一假设。超过2/3的重组者显示了模板切换，多股入侵或两者的明确证据。此外，我们发现了特定于跨界途径的活动的证据，包括分支迁移（分频器的2/3）和外核缝隙形成（交叉1/3）。当前的工作旨在确定负责这些活动的蛋白质。其他工作旨在通过使用一种新的方法来绘制霍利迪连接在重组中的位置来确认分支的迁移，我们开发了用于特异性净化含有霍利迪连接的中间体的新方法。最后，我们正在研究染色体结构如何，特别是减数分裂染色体轴，有助于调节重组。染色体轴成分（HOP1和RED1蛋白）的减数分裂特异性子集对减数分裂DSB的形成和伴侣选择很重要，并且相对于其他基因组的某些区域富集。使用一种新的方法将轴蛋白募集到通常耗尽这些蛋白质的区域，我们已经表明，高浓度的HOP1蛋白是减数分裂DSB形成的必要和足够的，但是这些DSB引发的重组事件并未遵循规范减数分裂的重组途径。我们目前正在确定HOP1促进DSB形成的机制，以及通过规范减数分裂重组机制修复Hop1依赖性DSB所需的其他因素。