Background: Eukaryotic DNA polymerase beta (pol beta), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. However, pol beta 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 beta (Danio pol beta) for biochemical characterization.
Results: Danio pol beta 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 beta 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 beta. The cDNA of Danio rerio pol beta encodes a protein of 337 amino acids, which is 2-3 amino acids longer than other pol beta proteins, and contains a P63D amino acid substitution, unlike mammalian pol beta s. This substitution lies in a hairpin sequence within an 8kDa domain, likely to be important in DNA binding. We performed extensive biochemical characterization of Danio pol beta in comparison with rat pol beta, which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30 degrees C). Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (k(cat)/K-m).
Conclusions: Our characterization of pol beta 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.