MECHANISM OF MEIOTIC RECOMBINATION INITATION IN YEAST

该项目的长期目标是了解减数分裂过程中同源重组启动的分子机制。重组对包括人类在内的大多数有性生殖生物的减数分裂是必不可少的，因为它产生了遗传多样性，并确保了同源染色体的准确分离。在酿酒酵母中，减数分裂重组通过Spo11催化的酯交换反应形成DNA双链断裂(DSB)，Spo11是一个广泛保守的家族成员，包括来自高等真核生物的减数分裂重组蛋白和考古细菌的II型拓扑异构酶。本研究将以Spo11为研究重点，探讨DSB形成的分子机制。具体目标是：L。识别与Spo11相互作用的蛋白质。除了Spo11外，DSB的形成至少还需要9种基因产物，这表明Spo11与其他蛋白质之间的物理相互作用对Spo11的功能非常重要。这些相互作用将使用几种互补的方法来鉴定：a)从减数分裂细胞的提取物中亲和纯化含有Spo11的复合体，并通过质谱学胰蛋白酶多肽指纹图谱鉴定蛋白质组分。B)DSB形成所需的蛋白质将在体外与Spo11共表达，并检测其形成复合体的能力。C)以污物为诱饵进行酵母菌双杂交筛选。2.鉴定SPORT的活性及其与Spo11相互作用的蛋白。Spo11将被过度表达和纯化，并将被检测DNA结合、DNA切割和拓扑异构酶活性。被发现与Spo11相互作用的蛋白质也将得到过度表达和纯化。将描述它们的活动及其对Spo11活动的影响。3.建立DSB形成的体外检测体系。最近的证据表明，染色质和减数分裂染色体的高阶结构影响双链断裂的位置和频率。为了开始解决这些效应的分子基础，将开发一种生物化学可及系统，该系统使用整个减数分裂染色体作为DSB形成的底物。已经确定了从野生型减数分裂细胞中分离的核在体外能够形成新的DSB的条件。该系统将进行优化和表征，然后通过分离野生型细胞核和适应从DSB缺陷突变细胞分离的核的体外完成来分离生化成分。

The long-term objective of this project is to understand the molecular mechanism of homologous recombination initiation during meiosis. Recombination is essential for meiosis in most sexually reproducing organisms, including humans, because it generates genetic diversity and ensures the accurate segregation of homologous chromosomes. In S. cerevisiae, meiotic recombination initiates via the formation of DNA double-strand breaks (DSBs) through a transesterification reaction catalyzed by Spo11, a member of a widely conserved family that includes meiotic recombination proteins from higher eukaryotes and type II topoisomerases of archaebacteria. This study will use Spo11 as the focal point to investigate the molecular mechanism of DSB formation. The Specific Aims are: l. To identify proteins that interact with Spo11. At least nine gene products in addition to Spo11 are required for DSB formation, suggesting that physical interactions between Spo11 and other proteins are important for Spo11 function. These interactions will be identified using several complementary approaches: a) Spo11-containing complexes will be affinity-purified from extracts of meiotic cells, and the protein components will be identified by mass spectrometric tryptic peptide fingerprinting. b) Proteins required for DSB formation will be coexpressed with Spo11 in vitro and assayed for complex formation. And c) yeast two-hybrid screens with Spoil as bait will be carried out. 2. To characterize the activities of Spoil and the proteins that interact with Spo11. Spo11 will be over-expressed and purified, and will be examined for DNA binding, DNA cleavage, and topoisomerase activities. Proteins found to interact with Spo11 will also be overexpressed and purified. Their activities and their effects on the activities of Spo11 will be characterized. 3. To develop an in vitro assay system for DSB formation. Recent evidence indicates that chromatin and higher order structures of meiotic chromosomes influence the locations and frequencies of DSBs. To begin to address the molecular basis of these effects, a biochemically accessible system that uses whole meiotic chromosomes as substrates for DSB formation will be developed. Conditions have been identified in which nuclei isolated from wild-type meiotic cells are competent to form new DSBs in vitro. This system will be optimized and characterized, then the biochemical components will be dissected by fractionation of wild- type nuclei, and by adaptation for in vitro complemenation of nuclei isolated from DSB-defective mutant cells.