Protein-primed replication of the bacteriophage PRD1 genome in vitro: Development of in vitro DNA replication system and characterization of replication origin.
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PublisherThe University of Arizona.
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AbstractA cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 (DNA polymerase) and 8 (terminal protein) of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-terminal protein complex as template, Mg²⁺, and four deoxyribonucleoside triphosphates. The origin and direction of PRD1 DNA replication in vitro were determined by restriction enzyme analysis of ³²P-labeled PRD1 DNA synthesized in this system. Replication starts at either end of the linear PRD1 DNA template. Analysis by alkaline sucrose gradient centrifugation and alkaline agarose gel electrophoresis of DNA synthesized in vitro showed that full-length PRD1 DNA is synthesized. DNA elongation in this system is inhibited by the drug aphidicolin. On the other hand, DNA initiation is inhibited by phenylglyoxal, an arginine-specific α-dicarbonyl reagent. In vitro studies have also demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. The minimal origin of replication was determined by analyzing the template activity of various deletion derivatives. It was shown that the terminal 20 bp of ITR are required for efficient in vitro DNA replication. It was found that, within the minimal replication origin region, there are complementary sequences which can form a small panhandle structure. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3' terminal C residue for the initiation of PRD1 DNA replication in vitro.
Degree ProgramMicrobiology and Immunology