Two family B DNA polymerases from Aeropyrum pernix, an aerobic hyperthermophilic crenarchaeote

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitiv e (fraction I) and aphidicolin-resistant (fraction II) activities were dete cted, The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family Il DNA polymerases were cloned from the organism by PCR using degenerated primers based on th e two conserved moths (motifA and B). The deduced amino acid sequences from their entire coding regions contained all of the moths identified in famil y B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up t o 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidico lin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal fa mily B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in eu ryarchaeotes, which contain one family B DNA polymerase and one heterodimer ic DNA polymerase of a novel family.