Affinity labeling of Escherichia coli DNA polymerase I by 5'-fluorosulfonylbenzoyladenosine. Identification of the domain essential for polymerization and Arg-682 as the site of reactivity.
Preincubation of Escherichia coli DNA polymerase I (pol I) with 5'-fluorosulfonylbenzoyladenosine (5'-FSBA) results in an irreversible inactivation of DNA polymerase activity with concomitant covalent binding of 5'-FSBA to enzyme. pol I-associated 3'-5' exonuclease activity, however, remains unaffected. Kinetic studies of inactivation indicate that the degree of inactivation is directly proportional to the concentration of 5'-FSBA and increases linearly with time. The presence of the metal chelate form of dNTP substrates or template primer, but not the template or primer alone, protects the enzyme from inactivation by 5'-FSBA. A complete inactivation of polymerase activity occurs when 2 mol of 5'-FSBA are covalently linked to 1 mol of enzyme, suggesting two sites of modification. Tryptic peptide mapping of 5'-FSBA-treated enzyme revealed the presence of two distinct peptides containing the affinity label, confirming the presence of two reactive sites in the enzyme. However, we find that only one of the two sites is essential for the polymerase activity since, in the presence of substrate dNTP or template primer during preincubation of enzyme with 5'-FSBA, incorporation of the affinity label is reduced by only 1 mol. Peptide analysis of dNTP or template primer-protected enzyme further revealed that a peptide eluting at 35 min from the C-18 matrix was protected from the 5'-FSBA reaction. It is therefore concluded that this peptide contains the domain essential for polymerase activity. Staphylococcus aureus V-8 protease digestion, amino acid composition, and sequence analysis of this peptide revealed this domain to span residues 669 to 687 in the primary amino acid sequence of pol I, and arginine 682 was found to be the site of 5'-FSBA reactivity.
Pandey
V N
VN
Department of Biochemistry, University of Medicine and Dentistry, New Jersey Medical School, Newark 07103.
Modak
M J
MJ
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism
D000345:Affinity Labels; D009711:Nucleotides; D011732:Pyridoxal Phosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D001120:Arginine; D004256:DNA Polymerase I; D000241:Adenosine