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


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.
Health Research Institute, National Institute of Advanced Industrial Science and Technology, Takamatsu, Kagawa, Japan.
Tomomi
T
Ishido
Naoshi
N
Yamazaki
Mitsuru
M
Ishikawa
Ken
K
Hirano
D000595:Amino Acid Sequence; D000818:Animals; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish / Q000235:genetics*
D029941:Fish Proteins; D011993:Recombinant Fusion Proteins; D019951:DNA Polymerase beta