Studies on mat1 Imprinting

mat1 印迹研究

• 批准号: 7965292
• 负责人: AMAR J KLAR
• 金额: $ 32.09万
• 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/7965292
• 关键词: Affect; Alkalies; Alleles; Binding; Biochemical; Biological Assay; Cell division; Cells; Centromere; Chromosomes; Complex; DNA; DNA Double Strand Break; DNA Modification Process; DNA biosynthesis; DNA purification; Development; Distal; Fission Yeast; Future; Gel; Genes; Genetic Screening; Goals; Mating Types; Modeling; Molecular; Molecular Analysis; Morphologic artifacts; Mutation; Pattern; Proteins; Replication Origin; Research Design; S cerevisiae SWI3 protein; Sister; Sister Chromatid; Site; Testing; Work; daughter cell; gene function; genetic pedigree; imprint; in vivo; novel; termination factor; two-dimensional

The pattern of switching of <I>S. pombe</i> 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 <I>mat1</i> imprinting that marks only one sister chromatid in a strand-specific manner, and that is related to site-specific, double-stranded DNA break at <I>mat1</i>. We now show that the imprint is a strand-specific, alkali-labile DNA modification at <I>mat1</i>.<BR><BR>The DNA break is an artifact, created from the imprint during DNA purification. We also proposed and tested the model that <I>mat1</i> 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 <I>mat1</i> or introducing an origin of replication, affects the imprinting and switching efficiencies in predicted ways. Two-dimensional gel analysis confirmed that <I>mat1</i> is preferentially replicated by a centromere-distal origin(s). Thus, the DNA replication machinery may confer different developmental potential to sister cells.<BR><BR> Our recent work has discovered biochemical functions of <I>swi1</i> and <I>swi3</i> genes. We found that swi1p and swi3p perform imprinting by pausing and termination of DNA replication at <I>mat1</i>. 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 <I>mat1</i>-proximal polar-terminator of replication (<I>RTS1</i>). 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 <I>mat1</i> and by terminating replication at <I>RTS1</i>. 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. We have defined a large number of mat1 mutations that affect imprinting. Their molecular analysis should help us define the mechanism of imprinting.

I S的转换模式。蓬贝> 当通过单细胞谱系测定时，细胞是非随机的。在连续两次不对称 在细胞分裂中，四分之一的“孙女”细胞经历交配型转换。在此之前， 我们证明了这种模式是由于I mat 1/i> 印迹，标记只有一个姐妹染色单体在一个链特异性的方式，这是 与位点特异性双链DNA断裂有关， <I mat1/i.我们现在表明，印记是一个 链特异性碱不稳定DNA修饰. <I mat1/i.& lt;BR BR The DNA断裂是一种人工制品，在DNA纯化过程中由印记产生。我们亦建议 并测试了I mat 1/i是 优先由着丝粒远端起源复制，使得链特异性 印迹仅在滞后链合成期间发生。改变复制起点， 求I mat 1/i的逆或引入 复制，以预测的方式影响印记和转换效率。 二维凝胶分析证实， <I mat 1/i优先由 着丝粒远端起源。因此，DNA复制机制可以赋予不同的 姐妹细胞的发育潜力。lt;BR BR> 我们最近的工作发现了 <我切换1/i， <I swi 3/i基因。我们发现swi 1 p和swi 3 p 通过暂停和终止DNA复制来进行印记， <I mat1/i.我们的研究表明：（1） swi 1 p和swi 3 p通过在印记位点暂停复制叉而起作用;以及（2）swi 1 p 和swi 3 p参与终止在 <I mat 1/i-近端极性-终止子 复制（I RTS 1/i）。基因筛查， 鉴定终止因子鉴定分离暂停/印记的等位基因， SWI 1 P功能的终止。结果表明，swi 1 p和swi 3 p促进印迹 以新颖的方式， <I mat 1/i，并通过终止复制 <I RTS 1/i.我们还发现Swi 1和Swi 3 蛋白质在体内形成复合物，并与RTS 1和mat 1复制暂停位点结合 在染色体上。未来的研究旨在确定印迹的机制。我们有 定义了大量影响印记的mat 1突变。他们的分子分析 应该能帮助我们定义印记的机制