Comparison of substrate specificities of Escherichia coli endonuclease IIIand its mouse homologue (mNTH1) using defined oligonucleotide substrates

Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen s pecies and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligo nucleotide substrates containing a urea residue (UR), two cis-thymine glyco l (TG) diastereoisomers, 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU ). The substrates were incubated with Endo III and mNTH1, and their activit ies were compared based on the product analysis by gel electrophoresis. End o III recognized all base lesions tested, but the activity for DHT was extr emely lower than other substrates. In contrast, albeit some preference of U R, mNTH1 showed essentially comparable activities for all substrates includ ing DHT. Comparison of the enzymatic parameters for cis-TG and DHT revealed that large decreases in the affinity (K-m, 27-fold) and k(cat) (11-fold) r elative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had c omparable affinities and k(cat) for both cis-TG and DHT, though turnover (k (cat)) of mNTH1 was notably slower than Endo III. In view of the reaction m echanism, the paired base effect on the damage recognition by the two enzym es was also examined. The activities of Endo III for UR and HOU were paired base-independent, but those for cis TG and DHT were significantly enhanced when paired with G. With mNTH1, the paired base effect was evident only fo r DHT. The variations of the repair activity with paired bases and enzymes are discussed in relation to the base flipping mechanism suggested for base excision repair enzymes.