Gene structure, purification and characterization of DNA polymerase β from Xiphophorus maculatus

• Published in: Comparative biochemistry and physiology. Toxicology & pharmacology 2004-07, Vol.138 (3), p.311-324
• Main Authors: Oehlers, Leon P., Heater, Sheila J., Rains, J. Douglas, Wells, Melissa C., David, Wendi M., Walter, Ronald B.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base excision repair; Cyprinodontiformes - genetics; DNA - biosynthesis; DNA Polymerase beta - chemistry; DNA Polymerase beta - genetics; DNA Polymerase beta - isolation & purification; DNA Polymerase beta - metabolism; DNA polymerase β; DNA Replication; DNA synthesis; Escherichia coli; Exons - genetics; Expression; Fidelity; Fish; Freshwater; Gene Expression; Humans; Introns - genetics; Kinetics; Molecular Sequence Data; Nucleotidyltransferase; Proofreading; Replication; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Xiphophorus; Xiphophorus maculatus
• ISSN: 1532-0456; 1878-1659
• DOI: 10.1016/j.cca.2004.06.003

Cloning of the Xiphophorus maculatus Polβ gene and overexpression of the recombinant Polβ protein has been performed. The organization of the XiphPolβ introns and exons, including intron–exon boundaries, have been assigned and were found to be similar to that for human Polβ with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase β (Polβ) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polβ, which catalyzed DNA synthesis on template–primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolβ for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polβ. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolβ and human Polβ were similar, although the recombinant Xiphophorus protein exhibited 2.5–7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polβ. In contrast, the recombinant XiphPolβ displayed significantly lower fidelities than human Polβ for dNTP insertion opposite a single-nucleotide gap at 37 °C.