DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [H-3]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 mu M and with an EC(50) (the effective concentration at 50% maximum photoincorporation) of about 0.74 mu M. Saturating concentrations of poly(dA).(T)(10) protect the Klenow fragment from [H-3]-1 photoincorporation, and TTP at a concentration approximately equal to its K-D for the free enzyme form shifts the dose-response curve for photoincorporation of [H-3]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [H-3]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 mu M in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp(732) in the peptide fragment Asp(732)-Ile(733)-His(734)-Arg(735) as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C-3'-exo and C-2'-exo-C-3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp(732) as an anchor point for the azide or-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.