Kee Kehkooi, Keeney Scott
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021, USA.
Genetics. 2002 Jan;160(1):111-22. doi: 10.1093/genetics/160.1.111.
In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.
在酿酒酵母中,引发减数分裂重组的DNA双链断裂(DSB)的形成至少需要10个基因的产物。Spo11p被认为是DNA切割活性的催化亚基,但其他蛋白质的作用以及它们之间的相互作用尚不清楚。本研究证明了SPO11的产物与另一个DSB形成所需的减数分裂早期基因REC102之间的遗传和物理相互作用。我们发现,单独能够支持正常或接近正常水平减数分裂重组的带有表位标签的SPO11和REC102版本,当在同一细胞中组合时会产生严重的合成冷敏感表型。在非允许温度下,双标签菌株中的DSB形成、减数分裂基因转换和孢子活力大幅降低。这种条件性缺陷可以通过未标记的SPO11的表达部分挽救,但不能通过未标记的REC102的表达挽救,这表明标记的REC102对这种合成表型完全显性。标记的和野生型的Spo11p都能与减数分裂细胞提取物中标记的Rec102p共免疫沉淀,表明这些蛋白质在体内存在于一个共同的复合物中。标记的Rec102p在全细胞中定位于细胞核,在铺展的减数分裂染色体上定位于染色质。我们的结果与这样一种观点一致,即包括Spo11p和Rec102p的多蛋白复合物促进减数分裂DSB的形成。