Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers

J Biol Chem. 2007 Jan 12;282(2):1098-108. doi: 10.1074/jbc.M606772200. Epub 2006 Nov 13.


Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276-21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Bacteriophage T4 / genetics
  • Bacteriophage T4 / growth & development*
  • Bacteriophage T4 / ultrastructure*
  • Biotin
  • DNA Helicases / chemistry
  • DNA Helicases / genetics
  • DNA Helicases / ultrastructure
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / ultrastructure
  • DNA, Viral / chemistry
  • DNA, Viral / ultrastructure
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / ultrastructure
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / ultrastructure
  • Microscopy, Electron, Transmission / methods*
  • Protein Structure, Quaternary
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / ultrastructure
  • Virus Replication*


  • DNA, Single-Stranded
  • DNA, Viral
  • DNA-Binding Proteins
  • Viral Proteins
  • gene 41 protein, Enterobacteria phage T4
  • gene 59 protein, Enterobacteria phage T4
  • gp32 protein, Enterobacteria phage T4
  • Biotin
  • DNA-Directed DNA Polymerase
  • DNA Helicases