Functional identification of an 8-oxoguanine specific endonuclease from Thermotoga maritima.
To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of NaBH4, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.
Im
Eun Kyoung
EK
Yonsei Research Institute of Aging Science, Yonsei University, Seoul 120-749, Korea.
Hong
Chang Hyung
CH
Back
Jung Ho
JH
Han
Ye Sun
YS
Chung
Ji Hyung
JH
D000595:Amino Acid Sequence; D002384:Catalysis; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004720:Endonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
D009841:Oligonucleotides; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004720:Endonucleases; D045647:DNA Glycosylases