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


Sandalli C., SINGH K., MODAK M. J. , KETKAR A., ÇANAKÇI S., DEMİR İ., ...Daha Fazla

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, cilt.84, sa.1, ss.105-117, 2009 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 84 Konu: 1
  • Basım Tarihi: 2009
  • Doi Numarası: 10.1007/s00253-009-1962-3
  • Dergi Adı: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • Sayfa Sayıları: ss.105-117

Özet

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'aEuro parts per thousand a dagger'aEuro parts per thousand 3' exonuclease activity, but lacks the motifs required for 3'aEuro parts per thousand a dagger'aEuro parts per thousand 5' exonuclease activity. 5' nuclease and 3'aEuro parts per thousand a dagger'aEuro parts per thousand 5' exonuclease assays confirmed that Gca polymerase I has a double-stranded DNA-dependent 5'aEuro parts per thousand a dagger'aEuro parts per thousand 3' nuclease activity but no 3'aEuro parts per thousand a dagger'aEuro parts per thousand 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 mu M, and 24.99 nucleotides(-1), respectively. The enzyme showed significant reverse-transcriptase activity (RT) with Mn2+, but very little RT activity with Mg2+. 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.