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

Abstract

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.

Comparison of DIG-11-dUTP utilization by Geobacillus caldoxylosilyticus TK4, Mycobacterium tuberculosis and Escherichia coli DNA polymerases

“…DNA-dependent DNA polymerase activity of these enzymes was shown by scintillation spectroscopy and primer extension assays (data not shown). Gca pol I (Sandalli et al 2009), Mtb pol I (Arrigo et al 2002) and KF are known so sensitive to ddNTP at different level and following its addition of this analogue into the DNA, the synthesis is blocked. Especially, Mtb pol I incorporates dideoxynucleotides more efficiently than KF and Gca pol I if the reaction mixture contains both dNTP and its ddNTP form of individual ddNTP.…”

“…The Gca pol I was expressed in E. coli and purified by performing heat treatment, DEAE-cellulose and BioRex 70 cationic chromatography (Sandalli et al 2009). The expression and purification of Mtb pol I enzyme was done according to Arrigo et al (2002).…”

“…5 0 exonuclease activity. They show also sensitivity to ddNTPs in different level (Sandalli et al 2009;Arrigo et al 2002).…”

Cloning and Sequence Analysis of Novel DNA Polymerases from Thermophilic Geobacillus Species Isolated from Hot Springs in Turkey: Characterization of a DNA Polymerase I from Geobacillus kaue Strain NB

“…The positions of amino acid residues were depicted based on BF structure [15,16] This is the second study, to our knowledge, a thermophilic family A DNA polymerase I from the genus Geobacillus was characterized biochemically. The first one was from DNA polymerase I from Geobacillus caldoxylosilyticus TK4 [14].…”

“…MKK [13], and Geobacillus caldoxylolyticus TK4 [14]. The crystal structure of a family A member DNA polymerase I from the genus Bacillus has only been identified for B. stearothermophilus DNA polymerase large fragment, known as Bacillus fragment (BF) [15,16].…”