A new DNA polymerase I from Geobacillus caldoxylosilyticus TK4: cloning, characterization, and mutational analysis of two aromatic residues.


DNA polymerase I gene was cloned and sequenced from the thermophilic ...
DNA polymerase I gene was cloned and sequenced from the thermophilic bacterium Geobacillus caldoxylosilyticus TK4. The gene is 2,634 bp long and encodes a protein of 878 amino acids in length. The enzyme has a molecular mass of 99 kDa and shows sequence homology with DNA polymerase I from Bacillus species (89% identity). The gene was overexpressed in Escherichia coli and the purified enzyme was biochemically characterized. It has all of the primary structural elements necessary for DNA polymerase and 5' --> 3' exonuclease activity, but lacks the motifs required for 3' --> 5' exonuclease activity. 5' nuclease and 3' --> 5' exonuclease assays confirmed that Gca polymerase I has a double-stranded DNA-dependent 5' --> 3' nuclease activity but no 3' --> 5' exonuclease activity. Its specific activity was observed to be 495,000 U/mg protein, and K (D) (DNA) , K (D) (dNTP) , and K (pol) were found to be 0.19 nM, 22.64 microM, and 24.99 nucleotides(-1), respectively. The enzyme showed significant reverse-transcriptase activity (RT) with Mn(2+), but very little RT activity with Mg(2+). Its error rate was found to be 2.5 x 10(-5) which is comparable to that of the previously reported error rate for the E. coli DNA polymerase I. Two aromatic residues required for dideoxyribonucleotide triphosphate sensitivity (F712Y) and strand displacement activity (Y721F) were identified.




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