MOLECULAR MECHANISMS OF S POMBE MEIOTIC RECOMBINATION

拟议研究的长期目标是确定 减数分裂同源重组的基本分子机制 裂殖酵母 具体目标是 1）分离和表征S. Pombe后期缺陷 减数分裂重组的步骤，如Holliday结构的解析 在同源染色体之间的减数分裂期间形成，以及2） 使用分子测定法确定S.粟属减数分裂重组 中间体和产品。 遗传学和生物化学方法 用于实现这些目标。 在遗传学方法中， 提出了一种识别S. Pombe突变体在晚期 减数分裂重组的步骤。 在这个屏幕上，一个突变体参与了一个 重组的后期步骤并产生不可存活的孢子， 一种额外的早期重组突变体表型， 交换开始。 在生物化学方法中，分子 测定用于检测和分析S.粟酒裂殖 重组中间体（例如，接合分子、缺口和双 链断裂）和成熟产物。 这些方法使用两个- 结合peptide的三维DNA分离测定 DNA的交联，引物延伸测定用于确定 含有霍利迪结构的中间体，以及分离的突变体 从前面提到的遗传学方法。 在真核生物中， 重组在以下方面发挥着核心作用：a）在两个区域产生多样性， 细胞和生物体水平，B）修复受损的染色体，和 c）减数分裂期间同源染色体的忠实分离。 重组畸变产生DNA突变（例如， 取代和缺失），和染色体重排（例如， 易位）和损失（例如，非整倍性）。 这种异常现象往往是 临床上表现为人类的癌症和出生缺陷。 S. pombe 在这里被用作真核生物的模型，以加深我们对 减数分裂重组的分子机制。 知识 在这里获得的将使用更多的了解到几个原因 人类遗传起源的疾病（例如，癌症），并可能导致 深入了解未来预防这些疾病的方法。

The long-term objective of the proposed research is to determine the basic molecular mechanism of meiotic homologous recombination in the fission yeast Schizosaccharomyces pombe. The specific aims are to 1) isolate and characterize mutants of S. Pombe defective in late steps of meiotic recombination such as resolution of Holliday structures formed during meiosis between homologous chromosomes and 2) determine, using molecular assays, S. Pombe meiotic recombination intermediates and products. Both genetic and biochemical approaches are used to achieve these objectives. In the genetic approach, a screen is proposed which will identify S. Pombe mutants defective in late steps of meiotic recombination. In this screen, a mutant involved in a late step of recombination and produces inviable spores is rescued by an additional early recombination mutant phenotype which prevents exchange from starting. In the biochemical approaches, molecular assays are used for detecting and analyzing S. pombe meiotic recombination intermediates (e.g., joint molecules, nicks, and double strand breaks) and mature products. These approaches use a two- dimensional DNA separation assay in combination with psoralen crosslinking of DNA, a primer extension assay for determining intermediates containing Holliday structures, and the mutants isolated from the genetic approach mentioned earlier. In eukaryotes, genetic recombination plays central roles in a) generating diversity at both the cellular and organismal levels, b) repair of damaged chromosomes, and c) faithful segregation of homologous chromosomes during meiosis. Recombinational aberrations generate DNA mutations (e.g., substitutions and deletions), and chromosome rearrangements (e.g., translocations) and loss (e.g., aneuploidy). Such aberrancies are often clinically manifested as cancer and birth defects in humans. S. pombe is used here as a model eukaryote to further our understanding of the molecular mechanisms of meiotic recombination. The knowledge gained here will give use more understanding into the causes of several diseases of genetic origin in humans (e.g., cancer) and possibly lead to insight into ways for preventing them in the future.