Abstract

The major aim of the proposed project is to get a better knowledge of the protein-primed mechanism of initiation of replication. We will study: 1) The specific sequence requirements at the 029 DNA ends for replication. 2) The interaction of the terminal protein-DNA polymerase complex with 029 DNA-protein p3 or with protein-free 029 DNA terminal fragments. 3) Protein p3 mutants will be constructed and used to determine the initiation activity and other properties of the protein. 4) Properties of the 029 DNA polymerase such as helicase activity, fidelity and its possible application in biotechnology will be studied. 5) Protein p2 mutants will be constructed and used to determine the initiation, elongation and 3' to 5' exonuclease activities and other properties of the protein. 6) The stimulation by protein p6 of the initiation and elongation steps of 029 DNA replication and the interaction with the 029 DNA ends will be studied to try to correlate these properties of the protein. 7) The role of protein p5 in 029 DNA replication will be determined. 8) The role of other viral proteins, such as pl and p17, in 029 DNA replication will be studied. 9) The role of transcription in 029 DNA replication and the role of protein p6 in 029 DNA transcription will be determined. 10) Replicative intermediates will be analyzed by electron microscopy to elucidate the way of replication of the displaced parental strand. 11) The role of the parental terminal protein in DNA encapsidation and the factors that determine the polarity in encapsidation will be studied. Health-related viruses such as adeno, polio, encephalomyocarditis, hepatitis A and B and viruses of socio- economic importance such as foot and mouth and several plant viruses, have a protein covalently linked to the 5' end(s) of the nucleic acid. Evidence for a protein-priming mechanism of replication has been obtained for 029 and adenovirus. There are indications that replication of poliovirus and encephalomyocarditis virus is initiated by a similar mechanism. The long term objective of the project will be to find specific ways to interfere with this new initiation reaction.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027242-11
Application #
3274643
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-01-01
Project End
1991-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Universidad Autonoma de Madrid
Department
Type
DUNS #
City
Madrid
State
Country
Spain
Zip Code

  Publications

Perez-Lago, Laura; Salas, Margarita; Camacho, Ana (2005) A precise DNA bend angle is essential for the function of the phage phi29 transcriptional regulator. Nucleic Acids Res 33:126-34
Asensio, Juan Luis; Albert, Armando; Munoz-Espin, Daniel et al. (2005) Structure of the functional domain of phi29 replication organizer: insights into oligomerization and dna binding. J Biol Chem 280:20730-9
Bravo, Alicia; Serrano-Heras, Gemma; Salas, Margarita (2005) Compartmentalization of prokaryotic DNA replication. FEMS Microbiol Rev 29:25-47
Meijer, Wilfried J J; Castilla-Llorente, Virginia; Villar, Laurentino et al. (2005) Molecular basis for the exploitation of spore formation as survival mechanism by virulent phage phi29. EMBO J 24:3647-57
Truniger, Veronica; Bonnin, Ana; Lazaro, Jose M et al. (2005) Involvement of the ""linker"" region between the exonuclease and polymerization domains of phi29 DNA polymerase in DNA and TP binding. Gene 348:89-99
Gonzalez-Huici, Victor; Alcorlo, Martin; Salas, Margarita et al. (2004) Phage phi29 proteins p1 and p17 are required for efficient binding of architectural protein p6 to viral DNA in vivo. J Bacteriol 186:8401-6
Kamtekar, Satwik; Berman, Andrea J; Wang, Jimin et al. (2004) Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29. Mol Cell 16:609-18
Gonzalez-Huici, Victor; Salas, Margarita; Hermoso, Jose M (2004) Genome wide, supercoiling-dependent in vivo binding of a viral protein involved in DNA replication and transcriptional control. Nucleic Acids Res 32:2306-14
Munoz-Espin, Daniel; Mateu, Mauricio G; Villar, Laurentino et al. (2004) Phage phi29 DNA replication organizer membrane protein p16.7 contains a coiled coil and a dimeric, homeodomain-related, functional domain. J Biol Chem 279:50437-45
Rodriguez, Irene; Lazaro, Jose M; Salas, Margarita et al. (2004) phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases. J Mol Biol 337:829-41

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