ENDONUCLEASE-III INTERACTIONS WITH DNA SUBSTRATES .2. THE DNA-REPAIR ENZYME ENDONUCLEASE-III BINDS DIFFERENTLY TO INTACT DNA AND TO APYRIMIDINIC APURINIC DNA SUBSTRATES AS SHOWN BY TRYPTOPHAN FLUORESCENCE QUENCHING

We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has two tryptophans, Trp(132) in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp(178) in the domain containing the iron-sulfur center of endonuclease III; Trp(132) is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., and Cunningham,:R. P. (1992) Science 258, 434-440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are,differently accessible to anionic quencher. Significant (similar to 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT). The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M(-1), and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was similar to 400-fold larger than to the undamaged 19-mer. Gel shift and footprinting experiments clearly show that duplex oligonucleotide substrates containing a central AP or noncatalyzable reduced AP site strongly bind to endonuclease III [O'Handley, S., Scholes, C. P., and Cunningham, R. P. (1995) Biochemistry 34, 2528-2536], but there was reduced fluorescence quenching in the presence of AP or reduced AP oligonucleotide substrates. The specific binding of such AP substrates must involve fluorescing tryptophan(s) differently from nonspecific binding to undamaged duplex DNA. This difference may correlate with the difference between the five- to seven-base pair size of the specific, AP-centered binding site determined by footprinting (companion paper) and the 12-base pair size of the nonspecific binding site determined from these fluorescence measurements. In competition with oligonucleotide 19-mer substrates containing either a central AP site or a reduced AP site, tryptophan fluorescence quenching brought on by high molecular weight poly(dAdT) DNA was eliminated and reduced to nearly its unquenched value. Quantitation of this competitive elimination indicated a specific binding constant of order 4 x 10(7) M(-1) for the reduced AP 19-mer substrate and a binding constant much greater than 10(8) M(-1) for the AP 19-mer substrate.