MECHANISM OF MEIOTIC RECOMBINATION INITATION IN YEAST

本项目的长期目标是了解减数分裂过程中同源重组起始的分子机制。重组对包括人类在内的大多数有性生殖生物的减数分裂至关重要，因为它产生遗传多样性并确保同源染色体的准确分离。在酿酒酵母中，减数分裂重组通过由Spo11催化的酯交换反应形成DNA双链断裂（DSBs）开始，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古细菌的II型拓扑异构酶。本研究将以Spo11为重点，探讨DSB形成的分子机制。具体目的是：1.鉴定与Spo11相互作用的蛋白质。除Spo11外，DSB的形成至少需要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的功能很重要。这些相互作用将通过几种互补的方法进行鉴定：a)含有spo11的复合物将从减数分裂细胞的提取物中亲和纯化，蛋白质成分将通过质谱色氨酸指纹图谱进行鉴定。b) DSB形成所需的蛋白将在体外与Spo11共表达，并检测是否形成复合物。c)以腐坏菌为诱饵进行酵母双杂交筛选。2. 表征Spo11的活性和与Spo11相互作用的蛋白质。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。发现与Spo11相互作用的蛋白质也会过度表达和纯化。本文将描述它们的活性及其对Spo11活性的影响。3. 建立DSB形成的体外检测系统。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dsb的位置和频率。为了开始解决这些影响的分子基础，将开发一种生物化学上可访问的系统，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细胞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缺陷突变细胞中分离的细胞核进行体外补体来解剖生化成分。

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.