Identification of 5‐formyluracil DNA glycosylase activity of human hNTH1 protein

• Published in: Nucleic acids research Vol. 30; no. 15; pp. 3443 - 3448
• Main Authors: Miyabe, Izumi, Zhang, Qiu‐Mei, Kino, Katsuhito, Sugiyama, Hiroshi, Takao, Masashi, Yasui, Akira, Yonei, Shuji
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.2002; Oxford Publishing Limited (England)
• Subjects: Deoxyribonuclease (Pyrimidine Dimer); DNA Glycosylases; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - metabolism; Escherichia coli Proteins; Humans; Kinetics; N-Glycosyl Hydrolases - chemistry; N-Glycosyl Hydrolases - metabolism; Oligonucleotides - chemistry; Oligonucleotides - metabolism; Schiff Bases - chemistry; Substrate Specificity; Uracil - analogs & derivatives; Uracil - chemistry; Uracil - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkf460

5‐Formyluracil (5‐foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The elucidation of repair mechanisms for 5‐foU will yield important insights into the biological consequences of the lesion. Recently, we reported that 5‐foU is recognized and removed from DNA by Escherichia coli enzymes Nth (endonuclease III), Nei (endonuclease VIII) and MutM (formamidopyrimidine DNA glycosylase). Human cells have been shown to have enzymatic activities that release 5‐foU from X‐ray‐irradiated DNA, but the molecular identities of these activities are not yet known. In this study, we demonstrate that human hNTH1 (endonuclease III homolog) has a DNA glycosylase/AP lyase activity that recognizes 5‐foU in DNA and removes it. hNTH1 cleaved 5‐foU‐containing duplex oligonucleotides via a β‐elimination reaction. It formed Schiff base intermediates with 5‐foU‐containing oligonucleotides. Furthermore, hNTH1 cleaved duplex oligonucleotides containing all of the 5‐foU/N pairs (N = G, A, T or C). The specific activities of hNTH1 for cleavage of oligonucleotides containing 5‐foU and thymine glycol were 0.011 and 0.045 nM/min/ng protein, respectively. These results indicate that hNTH1 has DNA glycosylase activity with the potential to recognize 5‐foU in DNA and remove it in human cells.