STUDIES ON MAT1 ON IMPRINTING

MAT1 印迹研究

• 批准号: 6423683
• 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/6423683
• 关键词: DNA damage; DNA replication origin; Schizosaccharomyces pombe; alleles; cell cycle; centromere; fungal genetics; genomic imprinting; site directed mutagenesis

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 artefact, 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.

当单细胞谱系分析时，pombe细胞的开关模式是非随机的。在连续两次不对称细胞分裂后，四分之一的后代细胞经历了交配型转换。先前，我们发现这种模式是由于mat1印迹仅以链特异性的方式标记一个姐妹染色单体，这与mat1位点特异性的双链DNA断裂有关。我们现在表明，印记是链特异性的，碱不稳定的DNA修饰在mat1。DNA断裂是一种人工制品，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源复制的模型，因此链特异性印记仅在滞后链合成期间发生。通过反转mat1或引入复制起始点来改变复制起始点，会以可预测的方式影响印迹和转换效率。二维凝胶分析证实，mat1优先由着丝粒远端起源复制。因此，DNA复制机制可能赋予姊妹细胞不同的发育潜能。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通过暂停和终止mat1上的DNA复制来执行印迹。我们的工作表明：(1)因子swi1p和swi3p通过在印迹位点暂停复制叉起作用；(2) swi1p和swi3p参与了mat1-proximal - polar-terminator of replication （RTS1）的终止。鉴定终止因子的遗传筛选发现了一个分离swi1p功能暂停/印记和终止的等位基因。结果表明，swi1p和swi3p通过暂停mat1位点的复制和终止RTS1位点的复制，以新颖的方式促进印迹。