Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus.
Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta 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 beta (Polbeta) 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 Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.
Oehlers
Leon P
LP
Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
Heater
Sheila J
SJ
Rains
J Douglas
JD
Wells
Melissa C
MC
David
Wendi M
WM
Walter
Ronald B
RB
D000595:Amino Acid Sequence; D000818:Animals; D003532:Cyprinodontiformes / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis; D019951:DNA Polymerase beta / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007438:Introns / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D004247:DNA; D019951:DNA Polymerase beta