Studies on mat1 Imprinting

mat1 印迹研究

• 批准号: 7053122
• 负责人: AMAR J KLAR
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7053122
• 关键词: DNA damage; DNA replication; DNA replication origin; Schizosaccharomyces pombe; alleles; cell cycle; centromere; fungal genetics; fungal proteins; gene expression; genetic screening; genomic imprinting; nucleic acid purification; protein binding; protein structure function; two dimensional gel electrophoresis

The pattern of switching of S. pombe cells is nonrandom when assayed by single cell pedigrees. After two consecutive asymmetric cell divisions, one in four "granddaughter" cells undergoes a mating-type switch. Previously, we showed that this pattern is due to mat1 imprinting that marks only one sister chromatid in a strand-specific manner, and that is related to site-specific, double-stranded DNA break at mat1. We now show that the imprint is a strand-specific, alkali-labile DNA modification at mat1. The DNA break is an artifact, created from the imprint during DNA purification. We also proposed and tested the model that mat1 is preferentially replicated by a centromere-distal origin(s), so that the strand-specific imprint occurs only during lagging-strand synthesis. Altering the origin of replication, by inverting mat1 or introducing an origin of replication, affects the imprinting and switching efficiencies in predicted ways. Two-dimensional gel analysis confirmed that mat1 is preferentially replicated by a centromere-distal origin(s). Thus, the DNA replication machinery may confer different developmental potential to sister cells. Our recent work has discovered biochemical functions of swi1 and swi3 genes. We found that swi1p and swi3p perform imprinting by pausing and termination of DNA replication at mat1. Our work shows (1) that the factors swi1p and swi3p act by pausing the replication fork at the imprinting site; and (2) that swi1p and swi3p are involved in termination at the mat1-proximal polar-terminator of replication (RTS1). A genetic screen to identify termination factors identified an allele that separated pausing/imprinting and termination of functions of swi1p. The results suggest that swi1p and swi3p promote imprinting in novel ways both by pausing replication at mat1 and by terminating replication at RTS1. We also showed that Swi1 and Swi3 proteins form a complex in vivo and both bind to the RTS1 and the mat1 replication pause sites on the chromosome. Future studies are designed to define the mechanism of imprinting

当通过单细胞谱系进行分析时，粟酒裂殖酵母细胞的转换模式是非随机的。在连续两次不对称细胞分裂后，四分之一的“孙女”细胞会经历交配型转换。之前，我们表明这种模式是由于 mat1 印记以链特异性方式仅标记一个姐妹染色单体，并且与 mat1 处的位点特异性双链 DNA 断裂有关。我们现在证明，印记是 mat1 上的链特异性、碱不稳定 DNA 修饰。 DNA 断裂是一种人工产物，是在 DNA 纯化过程中由印记产生的。我们还提出并测试了 mat1 优先由着丝粒远端起点复制的模型，因此链特异性印记仅在滞后链合成期间发生。通过反转 mat1 或引入复制起点来改变复制起点，会以预测的方式影响印记和转换效率。二维凝胶分析证实 mat1 优先由着丝粒远端复制。因此，DNA复制机制可能赋予姐妹细胞不同的发育潜力。 我们最近的工作发现了 swi1 和 swi3 基因的生化功能。我们发现 swi1p 和 swi3p 通过在 mat1 处暂停和终止 DNA 复制来进行印记。我们的工作表明（1）因子 swi1p 和 swi3p 通过在印记位点暂停复制叉来发挥作用； (2) swi1p 和 swi3p 参与 mat1 近端复制极性终止子 (RTS1) 的终止。鉴定终止因子的遗传筛选鉴定出一个等位基因，该等位基因将 swi1p 功能的暂停/印记和终止分开。结果表明 swi1p 和 swi3p 通过在 mat1 处暂停复制和在 RTS1 处终止复制以新颖的方式促进印记。我们还表明，Swi1 和 Swi3 蛋白在体内形成复合物，并且均与染色体上的 RTS1 和 mat1 复制暂停位点结合。未来的研究旨在定义印记机制