Characterization of the full length uracil-DNA glycosylase in the extreme thermophile Thermotoga maritima.
A full length (192 amino acids) uracil-DNA glycosylase (TMUDG) has been expressed and purified from the extreme thermophile Thermotoga maritima. This protein is active up to 85 degrees C. The enzyme is product inhibited by abasic sites in DNA and weakly inhibited by uracil. TMUDG was originally cloned from an ORF which encoded a protein of 185 amino acids. This shorter protein was stable up to 70-75 degrees C and it seemed unusual that this enzyme had an optimal activity temperature below the growth temperature of the organism (80-90 degrees C). Following the publication of the complete genomic sequence of T. maritima, it was shown that the gene contains an additional seven amino acids (LYTREEL) at the N-terminal end of the protein. It is suggested that these seven residues are important in maintaining proper protein folding that results in increased temperature stability. We have also demonstrated that TMUDG can substitute for the Escherichia coli uracil-DNA glycosylase and initiate base excision repair using a closed circular DNA substrate containing a unique U:G base pair.
Sandigursky
M
M
Departments of Radiology and Radiation Oncology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Faje
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Franklin
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D000595:Amino Acid Sequence; D016477:Artifacts; D002942:Circular Dichroism; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D051981:Uracil-DNA Glycosidase
D010446:Peptide Fragments; D011994:Recombinant Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase