A recombinant exonuclease III homologue of the thermophilic archaeon Methanothermobacter thermautotrophicus.
AP endonucleases catalyse an important step in the base excision repair (BER) pathway by incising the phosphodiester backbone of damaged DNA immediately 5' to an abasic site. Here, we report the cloning and expression of the 774 bp Mth0212 gene from the thermophilic archaeon Methanothermobacter thermautotrophicus, which codes for a putative AP endonuclease. The 30.3 kDa protein shares 30% sequence identity with exonuclease III (ExoIII) of Escherichia coli and 40% sequence identity with the human AP endonuclease Ape1. The gene was amplified from a culture sample and cloned into an expression vector. Using an E. coli host, the thermophilic protein could be produced and purified. Characterization of the enzymatic activity revealed strong binding and Mg2+-dependent nicking activity on undamaged double-stranded (ds) DNA at low ionic strength, even at temperatures below the optimum growth temperature of M. thermautotrophicus (65 degrees C). Additionally, a much faster nicking activity on AP site containing DNA was demonstrated. Unspecific incision of undamaged ds DNA was nearly inhibited at KCl concentration of approximately 0.5 M, whereas incision at AP sites was still complete at such salt concentrations. Nicked DNA was further degraded at temperatures above 50 degrees C, probably by an exonucleolytic activity of the enzyme, which was also found on recessed 3' ends of linearized ds DNA. The enzyme was active at temperatures up to 70 degrees C and, using circular dichroism spectroscopy, shown to denature at temperatures approaching 80 degrees C. Considering the high intracellular potassium ion concentration in M. thermautotrophicus, our results suggest that the characterized thermophilic enzyme acts as an AP endonuclease in vivo with similar activities as Ape1.
Pfeifer
Sven
S
Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig/Biotechnological-Biomedical Centre Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany.
Greiner-Stöffele
Thomas
T
D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D017124:Conserved Sequence; D017931:DNA Primers; D004260:DNA Repair; D004278:DNA, Superhelical / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017013:Methanobacteriaceae / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004278:DNA, Superhelical; D011994:Recombinant Proteins; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III