Mechanism of Meiotic Recombination

减数分裂重组机制

基本信息

批准号：
10262008
负责人：
MICHAEL J LICHTEN
金额：
$ 147.53万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10262008
关键词：
Biochemical Biological Models Biological Process Cell Cycle Arrest Cell Cycle Progression Chromosome Structures Chromosomes Complex Congenital Abnormality Cruciform DNA DNA Damage DNA Double Strand Break DNA Repair DNA damage checkpoint Defect Development Double Strand Break Repair Ensure Event Failure Genetic Genetic Recombination Genome Growth High-Throughput Nucleotide Sequencing Homologous Gene Human Infertility Lead Ligase Location Malignant Neoplasms Mediating Meiosis Meiotic Recombination Methods Mitosis Mitotic Cell Cycle Molecular Molecular Chaperones Mosaicism Pathway interactions Population Procedures Process Proteins Recombinants Regulation Repair Complex Research Role Saccharomyces cerevisiae Saccharomycetales Sister Chromatid Somatic Cell Testing Topoisomerase Work Yeasts base helicase homologous recombination insight member migration mutant novel prevent recruit repaired response tool topoisomerase IIIalpha transmission process

项目摘要

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. In addition, prompted by observations that mutants in the SUMO-ligase activity of the conserved Smc5-Smc6 DNA repair complex show meiotic recombination defects similar to STR complex mutants, we are testing the possibility that Smc5-Smc6-mediated SUMOylation of STR proteins is important for their meiotic activity. Studies of meiotic recombination in mutants lacking STR components also identified an Sgs1-independent role for Top3-Rmi1, in resolving strand entanglements that accumulate in recombination intermediates. Using a procedure call return-to-growth, where recombination intermediates are accumulated during wild-type meiosis and then resolved in a subsequent mitotic cell cycle, we have shown that Top3-Rmi1, but not Sgs1, is not required to fully resolve recombination intermediates. Unresolved recombination intermediates that remain in the absence of Top3-Rmi1 induce a DNA damage checkpoint that arrests cell cycle progression; we currently are determining how these intermediates are recognized and how they induce the DNA damage response. 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. We are currently determining the mechanism by which Hop1 promotes DSB formation, and if it impacts subsequent steps of meiotic recombination.

通过同源重组进行的减数分裂双链 DNA 断裂 (DSB) 修复通过由不同决策点定义的多个过程进行。一项重要的决定涉及伴侣的选择，是与姐妹染色单体（有丝分裂期间的显性修复伴侣）重组还是与同系物（不同亲本起源的同源染色体，减数分裂期间的首选伴侣）重组。另一个重要的决定涉及重组途径的选择，在产生交叉（交换侧翼染色体序列）或非交叉之间。我们小组的一个标志性贡献是证明交叉和非交叉重组通过不同的机制进行，这些机制在链入侵的初始阶段后出现分歧，并且具有不同的生化活性和遗传要求。之前审查期间的工作表明，保守的 Sgs1-Top3-Rmi1 解旋酶-拓扑异构酶复合物 (STR) 负责在非交叉和交叉途径之间划分早期重组事件。 Sgs1-Top3-Rmi1 是哺乳动物 BLM 解旋酶-Top3alpha-BLAP75 复合物的酵母同源物，与人类癌症避免和重组控制有关。我们表明，酵母复合体的所有三个成员对于正常重组伴侣的选择以及受调控的减数分裂交叉和非交叉重组途径的群体都是至关重要的。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分解，充当早期重组中间体的伴侣。我们假设这些链入侵和拆卸的重复循环将导致模板转换，这反过来又会导致具有嵌合亲本链贡献的重组体。这一假设现已通过在高度多态性测试间隔中发生的重组体高通量测序得到证实。超过 2/3 的重组体显示出模板转换、多链入侵或两者兼而有之的明确证据。此外，我们还发现了交叉途径特有活动的证据，包括分支迁移（交叉的 2/3）和核酸外切间隙形成（交叉的 1/3）。目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我们开发的专门纯化含有霍利迪连接体的中间体的新方法绘制重组中霍利迪连接体的位置来确认分支迁移。此外，由于观察到保守的 Smc5-Smc6 DNA 修复复合物的 SUMO 连接酶活性突变体显示出与 STR 复合体突变体相似的减数分裂重组缺陷，我们正在测试 Smc5-Smc6 介导的 STR 蛋白 SUMO 化是否重要的可能性因为它们的减数分裂活性。对缺乏 STR 成分的突变体减数分裂重组的研究也发现了 Top3-Rmi1 在解决重组中间体中积累的链缠结方面具有独立于 Sgs1 的作用。使用返回生长过程，其中重组中间体在野生型减数分裂期间积累，然后在随后的有丝分裂细胞周期中解决，我们已经证明，不需要 Top3-Rmi1 而不是 Sgs1 来完全解决重组中间体。在 Top3-Rmi1 缺失的情况下，未解析的重组中间体会诱导 DNA 损伤检查点，从而阻止细胞周期进程；我们目前正在确定如何识别这些中间体以及它们如何诱导 DNA 损伤反应。最后，我们正在研究染色体结构，特别是减数分裂染色体轴如何有助于重组的调节。染色体轴成分的减数分裂特异性子集 Hop1 和 Red1 蛋白对于减数分裂 DSB 形成和伴侣选择非常重要，并且相对于其他区域在基因组的某些区域中富集。使用一种新方法将轴蛋白募集到通常缺乏这些蛋白的区域，我们已经证明高浓度的 Hop1 蛋白对于减数分裂 DSB 的形成是必要且充分的。我们目前正在确定 Hop1 促进 DSB 形成的机制，以及它是否影响减数分裂重组的后续步骤。