Abstract

The objective of this project is to use bacteriophage PRD1 System, as a model, to investigate the mechanism of linear DNA replication. One of the major gaps in our knowledge of DNA replication is the mechanism by which the 5'-ends of 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. The genome of PRD1 is replicated by a protein-priming mechanism which preserves the 5'-end of the linear DNA. The PRD1 terminal protein (primer protein) and DNA polymerase are the smallest known proteins involved in linear DNA replication. For detailed structure-function analysis of these proteins, it is proposed that large quantities of the homogeneous terminal protein be prepared for crystallization, that sub- domain fragments be produced for solution NMR analysis and that site- specific mutants of terminal protein and DNA polymerase be constructed and analyzed.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM028013-12A2
Application #
2175067
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-01-01
Project End
1997-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
12
Fiscal Year
1994
Total Cost
Indirect Cost

  Institution

Name
University of Arizona
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721

  Publications

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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