title: Purification and characterization of PRD1 DNA polymerase.
abstract: A small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication. The purification of the PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in Escherichia coli cells. We have developed a simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase. This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E. coli containing the recombinant plasmid pEJG. The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties. These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis. Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude. The availability of a simple purification procedure for the PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724.
authors.forename: W
authors.initials: W
authors.lastname: Zhu
authors.forename: J
authors.initials: J
authors.lastname: Ito
mesh_terms: D003001:Cloning, Molecular; D003090:Coliphages / Q000201:enzymology* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase