Studies on mat1 Imprinting

开关&lt；I&gt；S的模式。当用单细胞谱系分析时，庞贝细胞是非随机的。在连续两次不对称细胞分裂后，四分之一的“孙女”细胞经历了交配型转换。先前，我们发现这种模式是由于&lt；I>mat1</ I&gt；印迹以链特异性的方式标记只有一个姐妹染色单体，这与位点特异性，双链DNA在&lt；I>mat1</ I&gt；处断裂有关。我们现在表明，印记是一种链特异性的、碱不稳定的DNA修饰，发生在&lt； 1</ I>;<;& gt;<；我们还提出并测试了一个模型，即&lt；I>mat1</ I&gt；优先由着丝粒远端起源复制，因此链特异性印记仅在滞后链合成期间发生。通过反转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，<I>mat1</ I&gt；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。我们最近的工作已经发现了&lt；I>swi1</ I&gt；和&lt；I>swi3</ I&gt；基因的生化功能。我们发现swi1p和swi3p通过在&lt；I>mat1</ I&gt；处暂停和终止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参与了复制的&lt；I>mat1</ I&gt； -近端极终止端（<I>RTS1</ I>）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通过在&lt；I>mat1</ I&gt；处暂停复制和在&lt；I>RTS1</ I&gt；处终止复制，以新颖的方式促进印迹。我们还发现Swi1和Swi3蛋白在体内形成一个复合体，并结合染色体上的RTS1和mat1复制暂停位点。未来的研究旨在明确印迹的机制。我们已经定义了大量影响印迹的mat1突变。它们的分子分析有助于我们确定印迹的机制。

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.