A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein

EMBO J. 2000 Jul 3;19(13):3418-27. doi: 10.1093/emboj/19.13.3418.

Abstract

We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.

Publication types

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

MeSH terms

  • Bacteriophage T7 / enzymology*
  • Base Sequence
  • DNA Primase / metabolism*
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism*
  • DNA, Viral / chemistry
  • DNA, Viral / metabolism*
  • Kinetics
  • Nucleic Acid Conformation
  • Protein Binding

Substances

  • DNA, Single-Stranded
  • DNA, Viral
  • DNA Primase