Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study.
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.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000345:Affinity Labels; D001665:Binding Sites; D004258:DNA Polymerase III / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D010777:Photochemistry; D013873:Thionucleotides / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000378:metabolism; D014466:Ultraviolet Rays
D000345:Affinity Labels; D003838:Deoxyadenine Nucleotides; D013873:Thionucleotides; D013942:Thymine Nucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate