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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Universidad Autonoma de Madrid
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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
Truniger, Veronica; Lazaro, Jose M; Salas, Margarita (2004) Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein binding. Nucleic Acids Res 32:361-70
Camacho, Ana; Salas, Margarita (2004) Molecular interplay between RNA polymerase and two transcriptional regulators in promoter switch. J Mol Biol 336:357-68
Gonzalez-Huici, Victor; Alcorlo, Martin; Salas, Margarita et al. (2004) Binding of phage Phi29 architectural protein p6 to the viral genome: evidence for topological restriction of the phage linear DNA. Nucleic Acids Res 32:3493-502
Truniger, Veronica; Lazaro, Jose M; Salas, Margarita (2004) Two positively charged residues of phi29 DNA polymerase, conserved in protein-primed DNA polymerases, are involved in stabilisation of the incoming nucleotide. J Mol Biol 335:481-94
Gonzalez-Huici, Victor; Alcorlo, Martin; Salas, Margarita et al. (2004) Bacteriophage O29 protein p6: an architectural protein involved in genome organization, replication and control of transcription. J Mol Recognit 17:390-6

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