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Background  Eukaryotic DNA polymerase β (pol β), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. However, pol β has not been purified or enzymatically characterized from the model fish species  Danio rerio  (zebrafish). We used the  in vitro / in vivo  dual expression system plasmid, pIVEX, to express  Danio rerio  pol β ( Danio  pol β) for biochemical characterization.    Results   Danio  pol β encoded by the  in vitro / in vivo -compatible pIVEX plasmid was expressed in  E. coli  BL21(DE3), BL21(DE3)pLysS, and KRX, and  in vitro  as a C-terminal His-tagged protein.  Danio  pol β expressed  in vitro  was subject to proteolysis; therefore, bacterial overexpression was used to produce the protein for kinetic analyses. KRX cells were preferred because of their reduced propensity for leaky expression of pol β. The cDNA of  Danio rerio  pol β encodes a protein of 337 amino acids, which is 2-3 amino acids longer than other pol β proteins, and contains a P63D amino acid substitution, unlike mammalian pol βs. This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. We performed extensive biochemical characterization of  Danio  pol β in comparison with rat pol β, which revealed its sensitivity to metal ion activators (Mn 2+  and Mg 2+ ), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30°C). Substituting Mn 2+  for Mg 2+  resulted in 8.6-fold higher catalytic efficiency ( k  cat / K  m ).    Conclusions  Our characterization of pol β from a model fish organism contributes to the study of the function and evolution of DNA polymerases, which are emerging as important cellular targets for chemical intervention in the development of anticancer agents.

Characterization of DNA polymerase β from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid