Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly

Biochemistry. 2005 Feb 22;44(7):2305-18. doi: 10.1021/bi0479508.


The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.

Publication types

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

MeSH terms

  • Bacteriophage T4 / enzymology*
  • Bacteriophage T4 / genetics
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / metabolism
  • DNA Helicases / chemistry*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism
  • DNA Primers / chemistry
  • DNA Primers / metabolism
  • DNA Replication* / genetics
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Exonucleases / antagonists & inhibitors
  • Exonucleases / metabolism
  • Fluorescence Resonance Energy Transfer / methods
  • Models, Molecular
  • Nucleic Acid Conformation
  • Nucleic Acid Heteroduplexes / chemistry
  • Nucleic Acid Heteroduplexes / metabolism
  • Nucleic Acid Synthesis Inhibitors
  • Protein Binding
  • Protein Interaction Mapping
  • Protein Processing, Post-Translational* / genetics
  • Replicon* / genetics
  • Substrate Specificity
  • Templates, Genetic
  • Viral Proteins / antagonists & inhibitors
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism


  • Cross-Linking Reagents
  • DNA Primers
  • DNA-Binding Proteins
  • Nucleic Acid Heteroduplexes
  • Nucleic Acid Synthesis Inhibitors
  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • gene 59 protein, Enterobacteria phage T4
  • DNA-Directed DNA Polymerase
  • Exonucleases
  • Deoxyribonucleases, Type II Site-Specific
  • GTAC-specific type II deoxyribonucleases
  • DNA Helicases