DNA-POLYMERASE PHOTOPROBE 2-[(4-AZIDOPHENACYL)THIO]-2'-DEOXYADENOSINE5'-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 th e 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 effect ive concentration at 50% maximum photoincorporation) of about 0.74 mu M. Saturating concentrations of poly(dA).(T)(10) protect the Klenow fr agment from [H-3]-1 photoincorporation, and TTP at a concentration app roximately equal to its K-D for the free enzyme form shifts the dose-r esponse curve for photoincorporation of [H-3]-1 into the Klenow fragme nt by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [H-3]-1 into the Kleno w fragment has an absolute requirement for magnesium, with no signific ant photoincorporation observed at concentrations of 1 up to 10 mu M i n the absence of magnesium. These results demonstrate that, as designe d, photoprobe 1 binds to both the dNTP and a portion of the template-p rimer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragmen t 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 ana lysis of the conformation of 1 indicated preferred C-3'-exo and C-2'-e xo-C-3'-endo symmetrical twist furanose ring puckers, with a high anti base conformation and a +sc C-5 torsional angle. Docking studies using Asp(732) as an anchor point for the azide or-nitrogen on the photolab el indicate that the dNTP binding site is at the edge of the DNA bindi ng cleft opposite the exonuclease site and that the template binding s ite includes helix O in the finger motif of the Klenow fragment.