Wallisch Michael, Kunkel Elisabeth, Hoehn Karsten, Grummt Friedrich
Institute of Biochemistry, University of Würzburg, Germany.
Biol Chem. 2002 May;383(5):765-71. doi: 10.1515/BC.2002.080.
A replication fork barrier at the 3'-end of mouse ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. This barrier is an inherent property of a defined DNA-protein complex including transcription termination factor I, and specific protein-protein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNA-binding protein is essential for barrier activity. We have purified and functionally characterised the protein from HeLa cells. The final preparation contained two polypeptides with molecular masses of 70 and 86 kDa, respectively. Both polypeptides interact with a GC-stretch adjacent to the binding site of transcription termination factor I. The specificity of binding to the barrier DNA was demonstrated in an electrophoretic mobility shift assay. The biochemical properties of this protein resemble that of Ku antigen, a human nuclear DNA-binding heterodimer that is the target of autoimmune-antibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from co-infected insect cells, is able to partially rescue the barrier activity in Ku-depleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent head-on collision between the replication and the transcription machinery.
小鼠核糖体RNA基因3'端的复制叉屏障可阻止双向叉的进展，并将DNA复制限制在与转录相同的方向。该屏障是一种特定DNA-蛋白质复合物的固有特性，该复合物包括转录终止因子I，并且在该因子与复制机制的蛋白质之间发生特定的蛋白质-蛋白质相互作用。在此，我们报道第二种DNA结合蛋白对于屏障活性至关重要。我们已从HeLa细胞中纯化并对该蛋白进行了功能表征。最终制备物包含分别具有70 kDa和86 kDa分子量的两种多肽。两种多肽均与转录终止因子I结合位点相邻的GC序列相互作用。在电泳迁移率变动分析中证明了与屏障DNA结合的特异性。该蛋白的生化特性类似于Ku抗原，Ku抗原是一种人类核DNA结合异二聚体，是几种自身免疫性疾病中自身抗体的靶标。从共感染的昆虫细胞中纯化的重组Ku蛋白作为异二聚体，能够部分挽救Ku缺失的HeLa细胞提取物中的屏障活性。这些数据表明，转录终止因子I和Ku协同作用以防止复制和转录机制之间的迎头碰撞。
