Nucleic Acids Research, 2001, Vol. 29, No. 3 604-613
© 2001 Oxford University Press
A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum
1Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, 2IGPP Center for Astrobiology, University of California, Los Angeles, CA 90095, USA and 3Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100°C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80–160 mM NaCl and 70°C, PaNth displays DNA glycosylase/ß-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
* To whom correspondence should be addressed at: Department of Microbiology and Molecular Genetics, 1602 Molecular Sciences Building, 405 Hilgard Avenue, Los Angeles, CA 90095, USA. Tel: +1 310 825 8460; Fax: +1 310 206 3088; Email: jhmiller{at}mbi.ucla.edu
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