Expression of human DNA polymerase beta in Escherichia coli and characterization of the recombinant enzyme.
The coding region of a human beta-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with beta-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as beta-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural beta-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant beta-polymerase using defined template-primer systems. The results indicate that this beta-polymerase is essentially identical with natural beta-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.
Abbotts
J
J
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
SenGupta
D N
DN
Zmudzka
B
B
Widen
S G
SG
Notario
V
V
Wilson
S H
SH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic
D011994:Recombinant Proteins; D004256:DNA Polymerase I