Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication

Nucleic Acids Res. 2007;35(21):7061-73. doi: 10.1093/nar/gkm749. Epub 2007 Oct 2.


To initiate phi29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related phi29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical 29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of phi29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus Phages / enzymology
  • Bacillus Phages / genetics*
  • Binding Sites
  • DNA Replication*
  • DNA, Viral / biosynthesis*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Replication Origin
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism


  • DNA, Viral
  • Recombinant Fusion Proteins
  • Viral Proteins
  • terminal protein, Bacillus phage phi29
  • DNA-Directed DNA Polymerase