Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.
Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, CA, 94143, USA.
Sci Rep. 2017 May 3;7(1):1393. doi: 10.1038/s41598-017-00742-3.
During Schizosaccharomyces pombe meiotic prophase, homologous chromosomes are co-aligned by linear elements (LinEs) analogous to the axial elements of the synaptonemal complex (SC) in other organisms. LinE proteins also promote the formation of meiotic DNA double-strand breaks (DSBs), the precursors of cross-overs. Rec10 is required for essentially all DSBs and recombination, and three others (Rec25, Rec27, and Mug20) are protein determinants of DSB hotspots - they bind DSB hotspots with high specificity and are required for DSB formation there. These four LinE proteins co-localize in the nucleus in an interdependent way, suggesting they form a complex. We used random mutagenesis to uncover recombination-deficient missense mutants with novel properties. Some missense mutations changed essential residues conserved among Schizosaccharomyces species. DSB formation, gene conversion, and crossing-over were coordinately reduced in the mutants tested. Based on our mutant analysis, we revised the rec27 open reading frame: the new start codon is in the previously annotated first intron. Genetic and fluorescence-microscopy assays indicated that the Rec10 N- and C-terminal regions have complex interactions with Rec25. These mutants are a valuable resource to elucidate further how LinE proteins and the related SCs of other species regulate meiotic DSB formation to form crossovers crucial for meiosis.
在裂殖酵母减数分裂前期，同源染色体通过类似于其他生物联会复合体（SC）的轴元件的线性元件（LinE）进行共排列。LinE 蛋白还促进减数分裂 DNA 双链断裂（DSB）的形成，这是交叉的前体。Rec10 对于基本上所有的 DSB 和重组都是必需的，另外三个（Rec25、Rec27 和 Mug20）是 DSB 热点的蛋白质决定因素——它们与 DSB 热点具有高度特异性结合，并且对于那里的 DSB 形成是必需的。这四种 LinE 蛋白以相互依赖的方式在核内共定位，表明它们形成了一个复合物。我们使用随机诱变来发现具有新特性的重组缺陷错义突变体。一些错义突变改变了在裂殖酵母物种中保守的必需残基。在测试的突变体中，DSB 形成、基因转换和交叉均协同减少。基于我们的突变分析，我们修订了 rec27 开放阅读框：新的起始密码子位于之前注释的第一个内含子中。遗传和荧光显微镜分析表明，Rec10 的 N 端和 C 端区域与 Rec25 具有复杂的相互作用。这些突变体是一个有价值的资源，可以进一步阐明 LinE 蛋白和其他物种的相关 SC 如何调节减数分裂 DSB 的形成以形成对减数分裂至关重要的交叉。
