Endonuclease III Interactions with DNA Substrates. 1. Binding and Footprinting Studies with Oligonucleotides Containing a Reduced Apyrimidinic Site

The binding of endonuclease III from Escherichia coli to damaged DNA has been studied using gel shift and footprinting assays. Oligonucleotides containing a reduced apyrimidinic (AP) site were used since reduction of the AP site blocks the β-elimination reaction catalyzed by the enzyme and yields a noncleavable substrate. The K_obs for a 13-mer carrying a centrally located reduced AP site is (2 x 10⁶)-(2 x 10⁷) M^-1, while the K_obs for a 13-mer with no damage is (4.5 x 10³)-(3.2 x 10⁴) M^-1 (approximately a 500-fold difference). Larger oligonucleotides would not enter a gel when endonuclease IH was bound so that binding constants to oligonucleotides longer than 13 base pairs could not be determined directly. Competition assays suggest that the K_obs measured for both damaged and undamaged 13-mers is a minimum value and that the K_obs for larger oligonucleotides could be an order of magnitude greater. Fluorescence quenching on related 19-mers yielded a specific binding constant for the 19-mer carrying a centrally located reduced AP site for 4 x 10⁷ M^-1 and a nonspecific binding constant to an undamaged 19-mer of approximately 10⁵ M^-1 [Xing, D., Dorr, R., Cunningham, R. P., & Scholes, C. P. (1995) Biochemistry 34, 2537-2544], Several footprinting reagents were used to determine the size and location of the endonuclease III binding site on damaged oligonucleotides. When endonuclease III is bound to a 25-mer or 39-mer duplex containing a centrally located reduced AP site, it protects 9-11 nucleotides on each strand from cleavage by deoxyribonuclease I and five nucleotides on each strand from cleavage by (methidiumpropyl-EDTA)Fe(II), indicating an extremely small binding site. Protection from hydroxyl radical cutting is only observed on the damaged strand, indicating that endonuclease III binds more tightly or closely to this strand. Also, endonuclease III does not protect guanine N7's in the major groove from methylation by dimethyl sulfate. Taken together, the footprinting studies imply that endonuclease III may bind along the minor groove side of the helix.