Studies on mat1 imprinting

对粟酒裂殖酵母（Schizoschiomycespombe，S.粟酒裂殖酵母）细胞在通过单细胞谱系测定时是非随机的。在两次连续的不对称细胞分裂后，四分之一的“孙女”细胞经历了交配型转换。以前，我们表明，这种模式是由于马特1印记，标志着只有一个姐妹染色单体在一个特定的方式，并与一个位点特异性，双链DNA断裂马特1。我们现在表明，这个印记是一个链特异性，碱不稳定的DNA修饰在mat 1。DNA断裂是一种人工制品，在DNA纯化过程中由印记产生。我们还提出和测试的模型，马特1优先复制的着丝粒远端的起源（S），使链特异性印记只发生在滞后链合成。改变复制起点，反转mat 1，或引入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，mat 1是优先复制的着丝粒远端的起源（S）。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swi 1和swi 3基因的生化功能。我们发现swi 1 p和swi 3 p通过在mat 1处暂停和终止DNA复制来执行印迹。我们的工作表明：1）因子swi 1 p和swi 3 p通过在印记位点暂停复制叉而起作用，2）swi 1 p和swi 3 p参与在复制的mat 1-近端极性终止子（RTS 1）处的终止。我们进行了遗传筛选，以确定这些终止因子，并确定了一个等位基因，分离的暂停/印记和终止功能的swi 1 p。我们的研究结果表明，swi 1 p和swi 3 p促进印迹在新的方式，无论是通过暂停复制在mat 1和终止复制在RTS 1。我们还表明，Swi 1和Swi 3蛋白在体内形成一个复合物，这两种蛋白质结合到RTS 1和染色体上的mat 1复制暂停位点。未来的研究将被设计来定义印迹的机制。我们已经发现了大量影响印记的mat 1突变。对这些mat 1突变的分子分析应该有助于我们确定印记的机制。

The pattern of switching of Schizosaccharomyces pombe (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 is related to a site-specific, double-stranded DNA break at mat1. We now show that this 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, 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 the biochemical functions of the swi1 and swi3 genes. We found that swi1p and swi3p perform imprinting by pausing and terminating DNA replication at mat1. Our work shows that: 1) the factors swi1p and swi3p act by pausing the replication fork at the imprinting site, and 2) swi1p and swi3p are involved in termination at the mat1-proximal polar-terminator of replication (RTS1). We performed a genetic screen to identify these termination factors and identified an allele that separated the pausing/imprinting and termination functions of swi1p. Our 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 that both proteins bind to the RTS1 and the mat1 replication pause sites on the chromosome. Future studies will be designed to define the mechanism of imprinting. We have already identified a large number of mat1 mutations that affect imprinting. Molecular analysis of these mat1 mutations should help us define the mechanism of imprinting.