Differentiation of the DnaA-oriC Subcomplex for DNA Unwinding in a Replication Initiation Complex

J Biol Chem. 2012 Oct 26;287(44):37458-71. doi: 10.1074/jbc.M112.372052. Epub 2012 Aug 31.


In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Amino Acid Substitution
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • DNA Footprinting
  • DNA Replication
  • DNA, Bacterial / chemistry*
  • DNA, Bacterial / genetics
  • DNA, Single-Stranded / chemistry
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Deoxyribonuclease I / chemistry
  • Electrophoretic Mobility Shift Assay
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Escherichia coli*
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Origin Recognition Complex / chemistry
  • Origin Recognition Complex / genetics*
  • Plasmids / genetics
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Structural Homology, Protein
  • Thermotoga maritima*
  • Transfection


  • Bacterial Proteins
  • DNA, Bacterial
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • DnaA protein, Bacteria
  • Escherichia coli Proteins
  • OriC chromosomal replication origin
  • Origin Recognition Complex
  • Adenosine Triphosphate
  • Deoxyribonuclease I