The polymerase domain of Streptococcus pneumoniae DNA polymerase I. High expression, purification and characterization.
The 3'-terminal two-thirds of the Streptococcus pneumoniae polA gene was cloned in an Escherichia coli genefusion vector with inducible expression. The resulting recombinant plasmid (pSM10) directs the hyperproduction of a polypeptide of 70.6 kDa corresponding to the C-terminal fragment of pneumococcal DNA polymerase I. Induced cells synthesized catalytically active protein to the extent of 7% of the total soluble protein in the cells. The polymerase fragment was purified to greater than 90% homogeneity with a yield of 1.5 mg pure protein/l culture. The protein has DNA polymerase activity, but no exonuclease activity. The enzyme requires a divalent cation (MgCl2 or MnCl2) for polymerization of DNA. Comparison of the mutant and wild-type pneumococcal polymerases shows that the construction did not affect the enzymatic affinity for the various substrates. The mutant protein, like its parent DNA polymerase I, exhibited an intermediate level of activity with primed single-stranded DNA. At high molar ratio of enzyme/DNA substrate, the polymerase fragment catalyzes strand displacement and switching after completing the replication of a primed single-stranded M13 DNA molecule.
Centro de Investigaciones Biológicas, Madrid, Spain.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics
D002413:Cations, Divalent; D010446:Peptide Fragments; D004256:DNA Polymerase I