Studies on Imat1I Imprinting

Imat1I印迹研究

• 批准号: 7592689
• 负责人: AMAR J KLAR
• 金额: $ 23.27万
• 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/7592689
• 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; Morphologic artifacts; Pattern; Proteins; Replication Origin; Research Design; S cerevisiae SWI3 protein; Sister; Sister Chromatid; Site; Testing; Work; daughter cell; 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.

<i> s的切换模式。当单细胞谱分析时，Pombe </i>细胞是非随机的。经过两个连续的不对称细胞分裂后，四分之一的“孙女”细胞经历了交配型开关。以前，我们证明了这种模式是由于<i> mat1 </i>烙印仅以特定于链特异性的方式标记一个姐妹染色单体，这与位点特异性的，双链的DNA断裂有关。现在，我们表明该烙印是一种特异性的，碱性的DNA修饰。我们还提出并测试了模型，即Mat1 </i>优先通过Centromere-Distal Origin（S）复制，因此仅在滞后链合成期间才会发生链特异性烙印。通过反转Mat1 </i>或引入复制的起源来改变复制的起源，以预测的方式影响印迹和切换效率。二维凝胶分析证实，<i> mat1 </i>优先通过Centromere-Distal Origin（S）复制。因此，DNA复制机制可能赋予姊妹细胞的不同发育潜力。<br> <br>我们最近的工作发现了<i> swi1 </i>和<i> swi3 </i>基因的生化功能。我们发现SWI1P和SWI3P通过在<i> mat1 </i>处暂停和终止DNA复制来执行印迹。我们的工作表明（1）SWI1P和SWI3P的因素通过在印迹地点暂停复制叉来起作用； （2）SWI1P和SWI3P在复制的<i> mat1 </i> - 二端极端终端参与了终止（<i> rts1 </i>）。识别终止因子的遗传筛选确定了一个等位基因，该等位基因将SWI1P功能的暂停/烙印和终止分开。结果表明，SWI1P和SWI3P通过在<i> mat1 </i>上暂停复制并通过以<i> rts1 </i>终止复制来促进新颖的印记。我们还表明，SWI1和SWI3蛋白在体内形成一个复杂的体内，并且都与染色体上的Mat1复制暂停位点结合。未来的研究旨在定义印迹的机制。