Bypass of a nick by the replisome of bacteriophage T7

J Biol Chem. 2011 Aug 12;286(32):28488-97. doi: 10.1074/jbc.M111.252023. Epub 2011 Jun 23.

Abstract

DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.

Publication types

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

MeSH terms

  • Bacteriophage T7 / chemistry
  • Bacteriophage T7 / genetics
  • Bacteriophage T7 / metabolism*
  • DNA Breaks, Single-Stranded*
  • DNA Helicases / chemistry
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA, Viral / biosynthesis*
  • DNA, Viral / chemistry
  • DNA, Viral / genetics
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism*
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • DNA, Viral
  • DNA-Binding Proteins
  • Multienzyme Complexes
  • Viral Proteins
  • DNA-Directed DNA Polymerase
  • DNA Helicases