The object of this project is to use bacteriophages Phi29 and PRD1 and model systems to investigate the mechanism of linear DNA replication. One of the major gaps of our knowledge of DNA replication is the mechanism by which the 5'-ends of the linear duplex DNA are replicated. Since none of the known DNA polymerases can initiate de novo synthesis of DNA chains, the elucidation of the primer forming mechanism is crucial to our understanding of DNA replication. Recently, novel DNA polymerases have been discovered which utilize protein as primers for the initiation of DNA chain synthesis. This project is designed for development of cell-free systems for complete semiconserved replication of linear DNA molecules of Phi29 and PRD1. Using these systems, a model will be tested which involves circular intermediate of DNA-protein complex and which accounts for replication of displaced strand. Both in vitro and in vivo crosslinking of DNA-protein complex will be investigated using electronmicroscopy and immunochemical methods. In addition, to understand details of protein-priming mechanism, it is proposed to isolate various mutants of DNA polymerases and DNA terminal proteins for both Phi29 and PRD1. It is proposed that mutant proteins be used for investigation of protein-protein, protein-DNA interactions and for structure-function analysis. If, as we believe, the basic mechanism of DNA replication may prove to be applicable to more complex systems, information obtained from these studies of bacterial viruses will undoubtedly contribute to our understanding of DNA replication in higher organisms.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Arizona
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Huang, Y P; Ito, J (1999) DNA polymerase C of the thermophilic bacterium Thermus aquaticus: classification and phylogenetic analysis of the family C DNA polymerases. J Mol Evol 48:756-69
Huang, Y P; Downie, J A; Ito, J (1999) Primary structure of the DNA polymerase I gene of an alpha-proteobacterium, Rhizobium leguminosarum, and comparison with other family A DNA polymerases. Curr Microbiol 38:355-9
Ito, J; Huang, Y; Parekh, T (1998) Studies on the cyanobacterial family C DNA polymerase. FEMS Microbiol Lett 158:39-43
Huang, Y P; Ito, J (1998) The hyperthermophilic bacterium Thermotoga maritima has two different classes of family C DNA polymerases: evolutionary implications. Nucleic Acids Res 26:5300-9
Cavallini, L; Coassin, M; Alexandre, A (1997) Diacylglycerol mediates the thrombin-induced, protein kinase C and Ca2+ independent activation of the Na+/H+ exchanger in platelets. FEBS Lett 400:99-102
Zhu, W; Ito, J (1994) Family A and family B DNA polymerases are structurally related: evolutionary implications. Nucleic Acids Res 22:5177-83
Braithwaite, D K; Ito, J (1993) Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res 21:787-802
Yoo, S K; Ito, J (1991) Sequence requirements for protein-primed DNA replication of bacteriophage PRD1. J Mol Biol 218:779-89
Ito, J; Braithwaite, D K (1991) Compilation and alignment of DNA polymerase sequences. Nucleic Acids Res 19:4045-57
Yoo, S K; Ito, J (1991) Initiation of bacteriophage PRD1 DNA replication on single-stranded templates. J Mol Biol 222:127-31

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