Nuclear factor I enhances adenovirus DNA replication by increasing the stability of a preinitiation complex.
Nuclear factor I (NFI) or its isolated DNA-binding domain (NFI-BD) enhances initiation of adenovirus DNA replication up to 50-fold at low concentrations of the precursor terminal protein-DNA polymerase (pTP-pol) complex. Both in solution and when bound to DNA, NFI-BD can form a complex with pTP-pol. To investigate the mechanism of enhancement by NFI, we determined the stability of a functional preinitiation complex formed in vitro between pTP-pol and the origin. Challenge experiments with a distinguishable template containing an identical origin revealed that in the absence of NFI, this preinitiation complex was very sensitive to competition for pTP-pol. Addition of NFI-BD increased the half-life of the complex at least 10-fold and led to the formation of a template-committed preinitiation complex. In agreement with this, binding of pTP-pol to origin DNA in band-shift assays was enhanced by NFI. By DNase I footprinting we show that the specificity of binding as well as induction of structural changes in origin DNA by pTP-pol are increased by NFI. These results indicate that NFI, by binding and positioning pTP-pol, stabilizes the complex between pTP-pol and the core origin, and thus enhances initiation of DNA replication.
Mul
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YM
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
Van der Vliet
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PC
D000260:Adenoviruses, Human / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I; D015684:Gene Products, pol / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008957:Models, Genetic; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015684:Gene Products, pol; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I