The Oct-1 POU domain stimulates adenovirus DNA replication by a direct interaction between the viral precursor terminal protein-DNA polymerase complex and the POU homeodomain

EMBO J. 1994 Nov 15;13(22):5401-9.

Abstract

The bipartite POU domain of transcription factor Oct-1 stimulates adenovirus DNA replication through an interaction with the octamer sequence present in the auxiliary origin. Employing an immobilized in vitro DNA replication system, we show that the POU domain enhances the formation of a pre-initiation complex composed of the viral precursor terminal protein-DNA polymerase (pTP-pol) complex and the origin. To investigate the mechanism of stimulation we have explored protein-protein interactions between the POU domain and the pTP-pol complex. Such an interaction could be detected using a GST-POU fusion protein bound to glutathione-agarose beads. Binding was also observed with the POU homeodomain (POUHD), albeit weaker than with the intact POU domain, but not with the POU specific subdomain. Four point mutations localized in the POUHD were analyzed for pTP-pol binding. Two of these, E22A and E30A, bound pTP-pol equally as well as the wild-type, while the other two, Q24A and E29A, were able to bind 2- to 4-fold better. These mutations are localized in the same region where the HSV transactivator VP16 binds, but did not coincide with the VP16 contacts. A direct correlation between pTP-pol binding and stimulation of DNA replication in vitro was observed for all mutants, suggesting that stimulation by the POU domain is caused by an interaction with the viral pTP-pol complex.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Adenoviridae / physiology*
  • Animals
  • Base Sequence
  • DNA Replication* / drug effects
  • DNA, Viral / genetics
  • DNA, Viral / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Herpes Simplex Virus Protein Vmw65 / metabolism
  • Host Cell Factor C1
  • Macromolecular Substances
  • Models, Molecular
  • Molecular Sequence Data
  • NFI Transcription Factors
  • Octamer Transcription Factor-1
  • Peptide Fragments / pharmacology
  • Point Mutation
  • Protein Binding
  • Protein Structure, Tertiary*
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Simplexvirus / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*
  • Viral Proteins / metabolism*
  • Virus Replication* / drug effects

Substances

  • DNA, Viral
  • DNA-Binding Proteins
  • Herpes Simplex Virus Protein Vmw65
  • Host Cell Factor C1
  • Macromolecular Substances
  • NFI Transcription Factors
  • Octamer Transcription Factor-1
  • Peptide Fragments
  • Pou2f1 protein, rat
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Viral Proteins
  • terminal protein, adenovirus
  • transcription factor nuclear factor 1
  • DNA-Directed DNA Polymerase