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。我们定义了大量影响印迹的MAT1突变。他们的分子分析应有助于我们定义印迹的机制。