Abstract
BackgroundEukaryotic 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.ResultsDanio 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 (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°C). Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (kcat/Km).ConclusionsOur 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.
Highlights
Eukaryotic DNA polymerase b, the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans
In the present study, we found pIVEX carrying the Danio rerio pol b (Danio pol b) cDNA causes growth inhibition of BL21(DE3)pLysS cells before expression induction, which contrasted with other proteins that were reported to be expressed in the pIVEX vector system using BL21(DE3)pLysS cells [19,20]
The cDNA of D. rerio pol b encodes a protein of 337 amino acids, which is two residues longer than the human, rat, calf, or mouse protein and three residues longer than that of frog (334 amino acids)
Summary
Eukaryotic DNA polymerase b (pol b), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. These pols have emerged as important cellular targets for chemical intervention in the development of anticancer agents [6,7] Of these pols, pol b is thought be involved in DNA repair synthesis [8]. Mammalian pol b is a single polypeptide of 39 kDa, comprising 335 amino acids forming two domains: an 8 kDa N-terminal domain and a 31 kDa C-terminal domain, connected with a protease-hypersensitive hinge region The former domain possesses a template-binding function and a dRPase activity to remove the 5’-deoxyribose phosphate, the latter domain possesses DNA polymerase activity. Biochemical studies have shown that pol b releases the 5’-terminal dRP residues from the aprinic/ apyrimidinic sites and fills short gaps of one to six nucleotides These activities indicate that pol b functions in base excision repair (BER), which is a major pathway for repairing modified bases in DNA [9]
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