INITIATION MECHANISM OF PROTEIN-LINKED DNA REPLICATION: THE FUNCTION OF BACILLUS SUBTILIS PHAGE PHI-29 TERMINAL PROTEIN.
KeywordsBacteriophages -- Physiological effect.
DNA -- Reproduction.
DNA -- Synthesis.
DNA viruses -- Physiological effect.
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PublisherThe University of Arizona.
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AbstractAn in vitro initiation of a DNA replication system was developed to study the function of Ø29 terminal protein. Cell free extracts prepared from Bacillus phage Ø29-infected cells catalyzed the formation of a complex between a 30,000 dalton protein and dAMP in the presence of MgCl₂, Ø29 DNA-protein template and α-³²P dATP. Uninfected cell extracts did not support this reaction. The molecular weight of this complex, the nature of linkage between dAMP and 30,000 dalton protein as well as nucleotide specificity for this reaction suggest that the protein moiety of this complex is the terminal protein of Ø29. Similar results were obtained with phages GA-1 and M2Y infected cell extracts. The similar requirements for complex formation and DNA replication in vitro implies that the complex formation is an initiation step in DNA replication. This notion was supported by characterizing the elongation product which formed in the presence of ddCTP. Two distinct antibodies were prepared for analyse the function of the terminal protein in Ø29 DNA replication. These antibodies react with native terminal protein as assayed by immunoprecipitation and ELISA methods. The inhibition of complex formation by these antibodies provides strong evidence that the terminal protein is involved in complex formation. The notion that complex formation is an initial step of DNA replication was demonstrated conclusively by inhibition of anti-TP on DNA replication in vitro. The results presented in this dissertation provide evidence supporting the protein-priming mode of linear Ø29 DNA replication.
Degree ProgramMolecular and Medical Microbiology