title: 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.
abstract: 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, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., & 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 (approximately 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 approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, University at Albany, State University of New York 12222.
authors.forename: D
authors.initials: D
authors.lastname: Xing
authors.forename: R
authors.initials: R
authors.lastname: Dorr
authors.forename: R P
authors.initials: RP
authors.lastname: Cunningham
authors.forename: C P
authors.initials: CP
authors.lastname: Scholes
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011067:Poly dA-dT / Q000378:metabolism; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011687:Purines; D011743:Pyrimidines; D011067:Poly dA-dT; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli