Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins.
The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.
Savilahti
H
H
Department of Genetics, University of Helsinki, Finland.
Caldentey
J
J
Lundström
K
K
Syväoja
J E
JE
Bamford
D H
DH
D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003854:Deoxyribonucleotides; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids
D004279:DNA, Viral; D003854:Deoxyribonucleotides; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases