Abstract

The major aim of the proposed project is to study the function in replication of protein p3, covalently linked to the 5' end of Bacillus subtilis phage 029 DNA. We will study: (a) The mechanism of the synthesis of the 029 DNA protein p3 complex. We will purify native protein p3 and study whether p3 itself has the enzymatic activity to catalyze the formation of the covalent bond with dAMP or some other phage-induced or bacterial activity is needed. The requirements in the template DNA and protein moieties will be also determined. (b) 029 DNA conformation required for replication. To investigate if circularization of 029 DNA in vivo has any role in replication we will repare DNA in which the protein at either one of the ends has been removed. After determining that this DNA is unable to circularize in vitro we will use it to transfect competent B. subtilis. (c) Possible role of transcription in DNA replication. The requirement for RNA polymerase activity or for the early promoters located close to the left and right DNA ends in the in vitro system of initiation of replication will be studied. (d) Cloning of 029 DNA fragments containing gene 3 in plasmids to overproduce protein p3. Bacteria transformed by these plasmids will be used as a starting material for the purification of protein p3. (e) Study of 029-induced enzymatic activities related to DNA replication and characterization of the genes coding for these activities by using sus and ts mutants in the genes involved in viral DNA synthesis. (f) Location of the serine residue in the protein involved in the linkage with the DNA, by identification and sequence determination of peptides containing the surrounding amino acids. (g) The role of protein p3 in DNA encapsulation and possible enzymatic activities in p3 (gyrase, ATPase) relevant to DNA packaging will be determined. Several health-related viruses such as adeno, hepatitis B, polio and encephalomyocarditis have a protein covalently linked to the 5' ends of the nucleic acid and a mechanism for the initiation of replication of these viruses similar to that of 029 has been proposed. The long-term objective of the project will be to find a specific way to interfere with this new initiation reaction once we understand its mechanism.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027242-06
Application #
3274640
Study Section
(MG)
Project Start
1980-01-01
Project End
1985-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
6
Fiscal Year
1985
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
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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