Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins

J Biol Chem. 1998 Feb 27;273(9):5260-70. doi: 10.1074/jbc.273.9.5260.


Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophage M13 / genetics*
  • Bacteriophage T7
  • Base Sequence
  • Biotinylation
  • DNA
  • DNA Helicases / metabolism
  • DNA Helicases / ultrastructure
  • DNA Primase / metabolism
  • DNA Primase / ultrastructure
  • DNA Primers
  • DNA Replication*
  • DNA, Circular / ultrastructure
  • DNA, Single-Stranded / ultrastructure
  • DNA, Viral / ultrastructure*
  • DNA-Binding Proteins / metabolism
  • DNA-Binding Proteins / ultrastructure
  • DNA-Directed DNA Polymerase / metabolism
  • DNA-Directed DNA Polymerase / ultrastructure
  • Deoxycytosine Nucleotides / chemistry
  • Deoxycytosine Nucleotides / metabolism
  • Molecular Sequence Data
  • Protein Binding
  • Viral Proteins / metabolism
  • Viral Proteins / ultrastructure


  • DNA Primers
  • DNA, Circular
  • DNA, Single-Stranded
  • DNA, Viral
  • DNA-Binding Proteins
  • Deoxycytosine Nucleotides
  • Okazaki fragments
  • Viral Proteins
  • gene 2.5 protein, Enterobacteria phage T7
  • 2'-deoxycytidine 5'-triphosphate
  • DNA
  • DNA Primase
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase
  • DNA Helicases