STUDIES ON MAT1 ON IMPRINTING

用单细胞家系分析时，蓬布链霉菌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，每四个孙子细胞中就有一个经历交配型转换。以前，我们证明这种模式是由于mat1印记，它只以链特异的方式标记一个姐妹染色单体，并与mat1处的位点特异性双链DNA断裂有关。我们现在证明印记是一种链特异性的，在Mat1处碱不稳定的DNA修饰。DNA断裂是一种人工制品，在DNA纯化过程中从印记中产生。我们还提出并测试了Mat1被着丝粒-远端起源(S)优先复制的模型，因此链特异性印记只发生在滞后链合成过程中。通过反转MAT1或引入复制起点来改变复制起点，以预测的方式影响印迹和切换效率。二维凝胶分析证实，mat1是由着丝粒-远端起源(S)优先复制的。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sw1和sw3基因的生化功能。我们发现SWIF1P和SWIF3P通过暂停和终止mat1处的DNA复制来执行印记。我们的工作表明：(1)SWIF1P和SWIF3P因子通过在印迹位点暂停复制叉来发挥作用；(2)SWIF1P和SWIF3P参与复制的mat1近端极终止子(RTS1)的终止。用于识别终止因子的遗传筛查确定了一个等位基因，该等位基因将sw1p的暂停/印记和功能终止分开。结果表明，SWIF1P和SWIF3P通过在mat1暂停复制和在RTS1终止复制，以新的方式促进印记。

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.