Gene structure, purification and characterization of DNA polymerase @b from Xiphophorus maculatus [An article from: Comparative Biochemistry and Physiology, Part C]

This digital document is a journal article from Comparative Biochemistry and Physiology, Part C, published by Elsevier in 2004. The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.

Description:
Cloning of the Xiphophorus maculatus Pol@b gene and overexpression of the recombinant Pol@b protein has been performed. The organization of the XiphPol@b introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Pol@b 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 @b (Pol@b) 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 Pol@b, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (K"m) and catalytic efficiencies (k"c"a"t/K"m) of the recombinant XiphPol@b for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Pol@b. Steady-state in vitro K"m and k"c"a"t/K"m values for correct nucleotide insertion by XiphPol@b and human Pol@b were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Pol@b. In contrast, the recombinant XiphPol@b displayed significantly lower fidelities than human Pol@b for dNTP insertion opposite a single-nucleotide gap at 37 ^oC.