Singh J, Goel V, Klar A J
Institute of Microbial Technology, Sector 39 A, Chandigarh 160 036, Punjab, India.
Mol Cell Biol. 1998 Sep;18(9):5511-22. doi: 10.1128/MCB.18.9.5511.
Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure.
最近的研究表明，在出芽酵母酿酒酵母中，DNA复制机制与交配型基因座的沉默相关联，并且在远缘相关的酵母粟酒裂殖酵母中可能存在类似的沉默机制。关于基因调控，DNA复制的一个重要功能可能是将忠实的染色质组装与子细胞中基因表达亲本状态的重建相耦合。我们一直致力于分离在这种假设的耦合中存在缺陷的突变体。从对温度敏感的生长和沉默表型的筛选中偶然分离出的一株粟酒裂殖酵母突变体，在沉默方面表现出一种新的缺陷，这种缺陷依赖于交配型基因座的转换能力，这一特性将该突变体与粟酒裂殖酵母以及酿酒酵母的其他沉默突变体区分开来。这种独特的突变体表型定义了一个基因座，我们将其命名为sng1（意为不受控制的沉默）。染色质分析显示，在sng1（-）突变体中，供体基因座mat2P和mat3M存在依赖于转换的解折叠，体内表达的大肠杆菌dam甲基化酶对其可及性增加表明了这一点。出乎意料的是，克隆和测序鉴定该基因是先前分离的DNA修复基因rhp6。酿酒酵母中的Rhp6同源物RAD6是复制后DNA修复以及组蛋白H2A和H2B泛素化所必需的。这项研究表明Rad6/rhp6蛋白参与基因调控，更重要的是，表明在染色体复制和重组过程中存在一个短暂的机会窗口来确保染色质结构的重塑。我们提出，sng1（-）/rhp6（-）突变对沉默的影响是染色质结构变化的间接后果。
