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

Abstract

The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of Gönen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5'-3' exonuclease domains, but the catalytic residues varied in 3'-5' exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5-8.5 in the presence of 8 mM Mg(2+) and 80-100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.

Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications

“…Enzymatic properties of the unmodified DNA polymerases I from genus Geobacillus have been [14][15][16][17][18][19][20], whereas this study is the first one, in which the biochemical characteristics of polymerases large fragments have been directly compared.…”

Molecular Cloning and Chartacterization DNA Polymerase I from thermophilic Geobacillus SBS 4S strain

“…Domain C is most important domain that is further divided into three subdomains designated as thumb, palm and fingers with highly conserved motifs A, B and C respectively (9). The palm subdomain has catalytic center and contains conserved carboxylate residues while subdomains fingers and thumb are involved into binding of DNA and incoming dNTPs during replication (10). Ten silent mutations were observed in polymerase I gene with one silent mutation in the C-terminal domain.…”

Physio-Biochemical Characterization and 16S rRNA Sequencing of Thermophilic Geobacillus sp. stain DB2 from Dibekli Hot Spring, Turkey

“…calidus ve Geobacillus thermodenitrificans subsp. thermodenitrificans (Cihan et al, 2011), Geobacillus kaue, Geobacillus anatolicus ve Geobacillus bogazici (Çağlayan and Bilgin 2011) Geobacillus galactosidasius DSM 18751 (Poli et al, 2011), Geobacillus thermoantarcticus DSM 9572 T (Coorevits et al, 2012), G. subterranaus subsp. aromaticivorans (Poli et al, 2012), Geobacillus icigianus (Bryanskaya et al, 2015) olarak databankta kayıtlıdır.…”