Mul Y M, Verrijzer C P, van der Vliet P C
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
J Virol. 1990 Nov;64(11):5510-8. doi: 10.1128/JVI.64.11.5510-5518.1990.
Initiation of adenovirus DNA replication is strongly enhanced by two transcription factors, nuclear factor I (NFI) and nuclear factor III (NFIII/oct-1). These proteins bind to two closely spaced recognition sequences in the origin. We produced NFI and NFIII/oct-1, as well as their biologically active, replication-competent DNA-binding domains (NFI-BD and the POU domain), in a vaccinia virus expression system and purified these polypeptides to apparent homogeneity. By DNase I footprinting and gel retardation, we show that the two proteins, as well as their purified DNA-binding domains, bind independently and without cooperative effects to their recognition sequences. By using a reconstituted system consisting of the purified viral proteins (precursor terminal protein-DNA polymerase complex (pTP-pol) and DNA-binding protein, we show that NFIII/oct-1 or the POU domain stimulates DNA replication in the absence of NFI or NFI-BD and vice versa. When added together, the enhancing effect of the two transcription factors was independent and nonsynergistic. Interestingly, stimulation by NFI or NFI-BD was strongly dependent on the concentration of the pTP-pol complex. At low pTP-pol concentrations, NFI or NFI-BD stimulated up to 50-fold, while at high concentrations, the stimulation was less than twofold, indicating that the need for NFI can be overcome by high pTP-pol concentrations. In contrast, stimulation by NFIII/oct-1 or the POU domain was much less dependent on the pTP-pol concentration. These data support a model in which NFI enhances initiation through an interaction with pTP-pol. Glutaraldehyde cross-linking experiments indicate contacts between pTP-pol and NFI but not NFIII/oct-1. The site of interaction is located in the NFI-BD domain.
腺病毒DNA复制的起始受到两种转录因子的强烈增强，即核因子I（NFI）和核因子III（NFIII/oct-1）。这些蛋白质与起始位点中两个紧密间隔的识别序列结合。我们在痘苗病毒表达系统中产生了NFI和NFIII/oct-1，以及它们具有生物学活性、能进行复制的DNA结合结构域（NFI-BD和POU结构域），并将这些多肽纯化至表观均一性。通过DNase I足迹法和凝胶阻滞实验，我们表明这两种蛋白质以及它们纯化的DNA结合结构域独立结合且无协同效应地与其识别序列结合。通过使用由纯化的病毒蛋白（前体末端蛋白-DNA聚合酶复合物（pTP-pol）和DNA结合蛋白）组成的重构系统，我们表明在没有NFI或NFI-BD的情况下，NFIII/oct-1或POU结构域能刺激DNA复制，反之亦然。当两者一起添加时，这两种转录因子的增强作用是独立且无协同性的。有趣的是，NFI或NFI-BD的刺激强烈依赖于pTP-pol复合物的浓度。在低pTP-pol浓度下，NFI或NFI-BD的刺激作用高达50倍，而在高浓度下，刺激作用小于两倍，这表明高pTP-pol浓度可以克服对NFI的需求。相比之下，NFIII/oct-1或POU结构域的刺激对pTP-pol浓度的依赖性要小得多。这些数据支持了一种模型，即NFI通过与pTP-pol相互作用来增强起始。戊二醛交联实验表明pTP-pol与NFI之间存在相互作用，但与NFIII/oct-1不存在相互作用。相互作用位点位于NFI-BD结构域。
