Abstract
Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen species and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligonucleotide substrates containing a urea residue (UR), two cis-thymine glycol (TG) diastereoisomers, 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU). The substrates were incubated with Endo III and mNTH1, and their activities were compared based on the product analysis by gel electrophoresis. Endo III recognized all base lesions tested, but the activity for DHT was extremely lower than other substrates. In contrast, albeit some preference of UR, mNTH1 showed essentially comparable activities for all substrates including 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) relative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had comparable 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 mechanism, the paired base effect on the damage recognition by the two enzymes 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 for 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.
| Original language | English |
|---|---|
| Pages (from-to) | 11389-11398 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 39 |
| Issue number | 37 |
| DOIs | |
| Publication status | Published - Sep 19 2000 |
| Externally published | Yes |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
Cite this
Comparison of substrate specificities of Escherichia coli endonuclease III and its mouse homologue (mNTH1) using defined oligonucleotide substrates. / Asagoshi, K.; Odawara, H.; Nakano, H.; Miyano, T.; Terato, Hiroaki; Ohyama, Y.; Seki, S.; Ide, H.
In: Biochemistry, Vol. 39, No. 37, 19.09.2000, p. 11389-11398.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Comparison of substrate specificities of Escherichia coli endonuclease III and its mouse homologue (mNTH1) using defined oligonucleotide substrates
AU - Asagoshi, K.
AU - Odawara, H.
AU - Nakano, H.
AU - Miyano, T.
AU - Terato, Hiroaki
AU - Ohyama, Y.
AU - Seki, S.
AU - Ide, H.
PY - 2000/9/19
Y1 - 2000/9/19
N2 - Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen species and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligonucleotide substrates containing a urea residue (UR), two cis-thymine glycol (TG) diastereoisomers, 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU). The substrates were incubated with Endo III and mNTH1, and their activities were compared based on the product analysis by gel electrophoresis. Endo III recognized all base lesions tested, but the activity for DHT was extremely lower than other substrates. In contrast, albeit some preference of UR, mNTH1 showed essentially comparable activities for all substrates including 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) relative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had comparable 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 mechanism, the paired base effect on the damage recognition by the two enzymes 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 for 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.
AB - Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen species and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligonucleotide substrates containing a urea residue (UR), two cis-thymine glycol (TG) diastereoisomers, 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU). The substrates were incubated with Endo III and mNTH1, and their activities were compared based on the product analysis by gel electrophoresis. Endo III recognized all base lesions tested, but the activity for DHT was extremely lower than other substrates. In contrast, albeit some preference of UR, mNTH1 showed essentially comparable activities for all substrates including 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) relative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had comparable 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 mechanism, the paired base effect on the damage recognition by the two enzymes 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 for 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.
UR - http://www.scopus.com/inward/record.url?scp=0034687125&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034687125&partnerID=8YFLogxK
U2 - 10.1021/bi000422l
DO - 10.1021/bi000422l
M3 - Article
VL - 39
SP - 11389
EP - 11398
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 37
ER -