Transcription factors NFI and NFIII/oct-1 function independently, employing different mechanisms to enhance adenovirus DNA replication.
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.
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Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
Verrijzer
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van der Vliet
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D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D051863:Host Cell Factor C1; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000502:physiology*; D050811:Octamer Transcription Factor-1; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000502:physiology*; D014779:Virus Replication
D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051863:Host Cell Factor C1; D046911:Macromolecular Substances; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D014157:Transcription Factors; D004259:DNA-Directed DNA Polymerase