Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study

J Biol Chem. 1984 Jun 25;259(12):7990-3.


The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique. ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP. No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP. ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites. Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit. Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide). This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.

Publication types

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

MeSH terms

  • Adenosine Diphosphate / pharmacology
  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Affinity Labels
  • Binding Sites
  • DNA Polymerase III / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Escherichia coli / enzymology*
  • Photochemistry
  • Thionucleotides / pharmacology
  • Thymine Nucleotides / metabolism
  • Ultraviolet Rays


  • Affinity Labels
  • Deoxyadenine Nucleotides
  • Thionucleotides
  • Thymine Nucleotides
  • adenosine 5'-O-(3-thiotriphosphate)
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase
  • 2'-deoxyadenosine triphosphate
  • thymidine 5'-triphosphate