BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS A new DNA polymerase I from Geobacillus caldoxylosilyticus TK4: cloning, characterization, and mutational analysis of two aromatic residues Cemal Sandalli & Kamalendra Singh & Mukund J. Modak & Amit Ketkar & Sabriye Canakci & İsmail Demir & Ali Osman Belduz Received: 8 January 2009 / Revised: 11 March 2009 / Accepted: 11 March 2009 / Published online: 14 April 2009 # Springer-Verlag 2009 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 biochemical- ly 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′ exonucle- ase 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 μM, 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 2.5×10 -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. Keywords Geobacillus caldoxylosilyticus TK4 . DNA polymerase I . Thermophilic . Mutation Introduction The integrity of all organisms depends on faithful genome replication. This is accomplished by DNA polymerases which synthesize DNA with extraordinary fidelity and efficiency to ensure proper transfer of genetic information from parent to progeny. For this purpose, all free-living organisms possess more than one kind of DNA poly- merases equipped with different functions. Based upon sequence comparison, DNA polymerases have been classi- fied into six families: A, B, C, D, X, and Y. The DNA polymerases that share sequence homology with Escher- ichia coli DNA polymerase I, II, and III have been assigned to the A, B, and C families, respectively. The DNA polymerases which did not show a sequence conservation with E. coli pol I, II, and III were grouped in X family (Delarue et al. 1990). Thus, mammalian DNA polymerase B belongs to the X family. Family D polymerases are thought to be replicative polymerases, and all known examples are found in Euryarchaeota of Archaea (Ishino et al. 1998). The newest class of DNA polymerases is the Y family which contains the DNA polymerases capable of lesion-bypass DNA synthesis (Ohmori et al. 2001; Hubscher et al. 2002). Appl Microbiol Biotechnol (2009) 84:105–117 DOI 10.1007/s00253-009-1962-3 C. Sandalli Department of Biology, Faculty of Arts & Sciences, Rize University, 53100 Rize, Turkey K. Singh : M. J. Modak : A. Ketkar Department of Biochemistry and Molecular Biology, UMD—New Jersey Medical School, Newark, NJ, USA S. Canakci : İ. Demir : A. O. Belduz (*) Department of Biology, Faculty of Arts & Sciences, Karadeniz Technical University, 6180 Trabzon, Turkey e-mail: belduz@ktu.edu.tr