Template primer-dependent binding of 5'-fluorosulfonyl-benzoyldeoxyadenosine by Escherichia coli DNA polymerase I. Identification of arginine 682 as the binding site and its implication in catalysis.
We have labeled a template primer-dependent substrate deoxynucleoside triphosphate binding domain in Escherichia coli DNA polymerase I using an affinity labeling analogue of dATP, the 5'-fluorosulfonylbenzoyldeoxyadenosine (FSBdA). Using enzyme-template primer complex as a test system, we find that FSBdA-mediated inactivation occurs only when the template in the enzyme-template primer complex is poly(dT).(dA)10. A ribonucleotide analogue, 5'-fluorosulfonylbenzoyladenosine (FSBA) is not an effective inactivator under these conditions. In the absence of template primer, however, deoxyribonanalogue (FSBdA) irreversibly inactivates polymerase activity with characteristics similar to those reported for FSBA (Pandey, V.N., and Modak, M.J. (1988) J. Biol. Chem. 263, 6068). Binding stoichiometric studies in the presence and absence of template primer revealed that only 1 mol of FSBdA is incorporated per mol of enzyme which results in complete inactivation. The site of FSBdA action was investigated by comparative tryptic peptide mapping, followed by amino acid composition analysis of the modified peptide. Arginine 682 was found to be the target of FSBdA reactivity. We therefore conclude that the domain containing Arg-682 plays a major role in template-dependent dNTP binding and polymerization.
Pandey
V N
VN
Biochemistry Division, Bhabha Atomic Research Center, Bombay, India.
Kaushik
N A
NA
Pradhan
D S
DS
Modak
M J
MJ
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003839:Deoxyadenosines / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013698:Templates, Genetic; D014357:Trypsin
D000345:Affinity Labels; D003839:Deoxyadenosines; D010446:Peptide Fragments; C063171:5'-fluorosulfonylbenzoyldeoxyadenosine; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D001120:Arginine; D004256:DNA Polymerase I; D014357:Trypsin; D000241:Adenosine