Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.
The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.
Chen
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M
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Mermod
N
N
Horwitz
M S
MS
D000260:Adenoviruses, Human / Q000201:enzymology / Q000502:physiology*; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D011233:Precipitin Tests; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors; D014764:Viral Proteins / Q000378:metabolism; D016335:Zinc Fingers
D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D051839:NFI Transcription Factors; D014157:Transcription Factors; D014764:Viral Proteins; C056461:terminal protein, adenovirus; D004259:DNA-Directed DNA Polymerase