Sap1p binds to Ter1 at the ribosomal DNA of Schizosaccharomyces pombe and causes polar replication fork arrest
Sap1p binds to Ter1 at the ribosomal DNA of Schizosaccharomyces pombe and causes polar replication fork arrest
复制标题
DOI：
10.1074/jbc.m508996200
复制
发表时间：
2005-11-25
影响因子：
4.8
通讯作者：
Bastia, D
中科院分区：
文献类型：
--
作者：
Krings, G;Bastia, D
在未受干扰的DNA复制过程中，真核生物的DNA复制叉在位于核糖体DNA(RDNA)基因间隔区内的自然复制叉障碍或Ter位置停滞。裂殖酵母裂殖酵母rDNA基因间隔区含有四个极性或定向特异的分叉障碍：Ter1-3和RFP4。虽然转录终止子Reb1p与终端2和终端3结合以阻止复制，但导致终端1和RFP4分叉阻止的因子(S)仍不清楚。使用接头扫描突变，我们已经将最小的Ter1缩小到21个碱基对。序列分析表明，在该区域内存在一个基本的开关激活和基因组稳定蛋白Sap1p的共识结合基序。重组Sap1p与Ter1结合具有很高的特异性，内源性Ter1结合活性含有Sap1p，并与Sap1p-Ter1复合体共迁移。循环排列分析表明，Sap1p在结合时使Ter1和SAS1弯曲。靶向突变分析表明，在体外阻止Sap1p结合的Ter1突变在体内的复制分叉抑制方面存在缺陷，而不影响Sap1p结合的突变仍然能够阻止复制。结果证实了染色质组织者Sap1p与SAS1以外的基因组区域特异性结合的假设，并支持Sap1p与rDNA分叉屏障Ter1结合导致该位点而不是SAS1处的极性复制分叉停滞的观点。
Eukaryotic DNA replication forks stall at natural replication fork barriers or Ter sites located within the ribosomal DNA ( rDNA) intergenic spacer regions during unperturbed DNA replication. The rDNA intergenic spacer of the fission yeast Schizosaccharomyces pombe contains four polar or orientation-specific fork barriers, Ter1-3 and RFP4. Whereas the transcription terminator Reb1p binds Ter2 and Ter3 to arrest replication, the factor(s) responsible for fork arrest at Ter1 and RFP4 remain unknown. Using linker scanning mutagenesis, we have narrowed down minimal Ter1 to 21 bp. Sequence analysis revealed the presence of a consensus binding motif for the essential switch-activating and genome-stabilizing protein Sap1p within this region. Recombinant Sap1p bound Ter1 with high specificity, and endogenous Ter1 binding activity contained Sap1p and comigrated with the Sap1p-Ter1 complex. Circular permutation analysis suggested that Sap1p bends Ter1 and SAS1 upon binding. Targeted mutational analysis revealed that Ter1 mutations, which prevent Sap1p binding in vitro, are defective for replication fork arrest in vivo, whereas mutations that do not affect Sap1p binding remain competent to arrest replication. The results confirm the hypothesis that the chromatin organizer Sap1p binds site-specifically to genomic regions other than SAS1 and support the notion that Sap1p binds the rDNA fork barrier Ter1 to cause polar replication fork arrest at this site but not at SAS1.