Structural Analyses of gp45 Sliding Clamp Interactions During Assembly of the Bacteriophage T4 DNA Polymerase Holoenzyme. I. Conformational Changes Within the gp44/62-gp45-ATP Complex During Clamp Loading

J Biol Chem. 1997 Dec 12;272(50):31666-76. doi: 10.1074/jbc.272.50.31666.

Abstract

A multisubunit ring-shaped protein complex is used to tether the polymerase to the DNA at the primer-template junction in most DNA replication systems. This "sliding clamp" interacts with the polymerase, completely encircles the DNA duplex, and is assembled onto the DNA by a specific clamp loading complex in an ATP-driven process. Site-specific mutagenesis has been used to introduce single cysteine residues as reactive sites for adduct formation within each of the three subunits of the bacteriophage T4-coded sliding clamp complex (gp45). Two such mutants, gp45S19C and gp45K81C, are reacted with the cysteine-specific photoactivable cross-linker TFPAM-3 and used to track the changes in the relative positioning of the gp45 subunits with one another and with the other components of the clamp loading complex (gp44/62) in the various stages of the loading process. Cross-linking interactions performed in the presence of nucleotide cofactors show that ATP binding and hydrolysis, interaction with primer-template DNA, and release of ADP all result in significant conformational changes within the clamp loading cycle. A structural model is presented to account for the observed rearrangements of intersubunit contacts within the complex during the loading process.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Binding Sites
  • Cross-Linking Reagents / pharmacology
  • DNA Replication*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Fluorobenzenes / pharmacology
  • Macromolecular Substances
  • Maleimides / pharmacology
  • Models, Chemical
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Photochemistry
  • Protein Conformation
  • T-Phages / enzymology*
  • Trans-Activators / chemistry
  • Trans-Activators / metabolism*
  • Viral Proteins / chemistry
  • Viral Proteins / metabolism*

Substances

  • Cross-Linking Reagents
  • Fluorobenzenes
  • Macromolecular Substances
  • Maleimides
  • T7C-45 protein, Bacteriophage T4
  • Trans-Activators
  • Viral Proteins
  • gene 44 protein, Enterobacteria phage T4
  • gene 45 protein, Enterobacteria phage T4
  • gp62 protein, bacteriophage T4
  • N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide
  • Adenosine Triphosphate
  • DNA-Directed DNA Polymerase