Nuclear factor I enhances adenovirus DNA replication by increasing the stability of a preinitiation complex

EMBO J. 1992 Feb;11(2):751-60.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviruses, Human / genetics*
  • CCAAT-Enhancer-Binding Proteins*
  • DNA Replication*
  • DNA, Viral / genetics*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonuclease I
  • Gene Products, pol / genetics
  • Gene Products, pol / metabolism
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Kinetics
  • Models, Genetic
  • NFI Transcription Factors
  • Nuclear Proteins
  • Templates, Genetic
  • Transcription Factors / metabolism*
  • Vaccinia virus / genetics
  • Y-Box-Binding Protein 1

Substances

  • CCAAT-Enhancer-Binding Proteins
  • DNA, Viral
  • DNA-Binding Proteins
  • Gene Products, pol
  • NFI Transcription Factors
  • Nuclear Proteins
  • Transcription Factors
  • Y-Box-Binding Protein 1
  • YBX1 protein, human
  • DNA-Directed DNA Polymerase
  • Deoxyribonuclease I