Wednesday, 23rd January 2019

Genome Dynamics and Function

     Replication of Bacteriophage ø29 DNA

 

 

Grupo

 


Margarita Salas

 

ESciStaff

 

EPublications

 

 

 

Research summary:

We have continued with the study of ø29 DNA replication initiated by TP-priming. We have dissected the role of the TP DNA binding residues in the replication of the viral DNA. Also, we have determined that the aromatic residue Phe230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerization active site to direct insertion of the initiator dAMP. We have identified one site in the TP that allows the insertion of peptides up to 17 amino acids while maintaining the ability to support DNA amplification in vitro. In relation to the ø29 DNA polymerase, in collaboration with Dr. Borja Ibarra, we have determined the mechanism of translocation during processive DNA replication. Besides, we have carried out a global transcriptional analysis of virus-host interactions between ø29 and Bacillus subtilis, finding genes that are up-regulated and others that are down-regulated. On the other hand, we have purified and characterized the DNA polymerase of phage Bam35, that infects Bacillus thuringiensis, with the finding that it is a very faithful polymerase that can couple strand displacement to processive DNA synthesis. In addition, it is able to perform abasic sites translesion synthesis. We have also characterized the Bam35 TP and shown that it is used as primer in the initiation of replication of the viral DNA. In collaboration with Dr. Nadine Fornelos, we have shown that protein gp7 of the B. thuringiensis phage GIL01, regulates transcription interacting with the bacterial LexA repressor.

We have generated a recombinant of African swine fever virus (ASFV), BA71 isolate, that is deleted in the viral gene CD2v, homologous to the celular CD2. This recombinant, designated BA71∆CD2, is highly attenuated in vivo and has demonstrated to confer very solid protection against experimental challenge with lethal homologous and heterologous ASF viruses.

 

figure 1

Fig. 1: Bacteriophage Bam35 as a new working model for protein-primed DNA replication: early steps of TP-DNA replication. A novel single-nucleotide jumping back is involved in Bam35 genome replication (middle row), in comparison with ø29 sliding back and adenovirus jumping back.

 


 

Publications:

  • Mojardín, L., Botet, J., Moreno, S. and Salas, M. (2015). Chromosome segregation and organization are targets of 5´-Fluorouracil in eukaryotic cells. Cell Cycle 14, 206-218.
  • Holguera, I., Muñoz-Espín, D. and Salas, M. (2015). Dissecting the role of DNA-binding residues of the ø29 terminal protein in viral DNA replication. Nucleic Acids Res. 43, 2790-801.
  • Morin, A,. Cao, F.J., Lázaro, J. M., Arias-Gonzalez, J.R., Valpuesta, J. M., Carrascosa, J.L., Salas, M. and Ibarra, B. (2015). Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase. Nucleic Acids Res. 43, 3643-3652.
  • Köhler, K., Duchardt-Fener, E., Lechner, M., Damm, K., Hoch, P.G., Salas, M. and Hartmann, R.K. (2015). Structural and mechanistic characterization of 6S RNA from the hyperthermophilic bacterium Aquifex aeolicus. Biochimie 117, 72-86.
  • Berjón-Otero, M., Villar, L., de Vega, M., Salas, M. and Redrejo-Rodríguez, M. (2015). DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity. Proc.Natl.Acad.Sci.USA. 112, 3476-3484.
  • Fornelos, N., Butala, M., Hodnik, V., Anderluh, G., Bamford, J. K. and Salas, M. (2015). Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage. Nucleic Acids Res. 43, 7315-7319.
  • del Prado, A., Lázaro, J.M., Longás, E., Villar, L., de Vega M. and Salas, M. (2015). Insights into the determination of the templating nucleotide at the initiation of 29 DNA replication. J.Biol.Chem. 290, 27138-27145.
  • Salas, M., Holguera, I., Redrejo-Rodríguez, M. and de Vega, M. (2016). DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication. Frontiers in Molecular Biosciences. 3, 37.
  • Berjón-Otero, M., Villar, L., Salas, M. and Redrejo-Rodríguez, M. (2016). Disclosing early steps of protein-primed genome replication of the Grampositive tectivirus Bam35. Nucleic Acids Res. 44, 9733-9744.
  • Mojardín L. and Salas, M. (2016). Global transcriptional analysis of virus-host interactions between phage ø29 and Bacillus subtilis. J. Virol. 90, 9293-9304.
  • Gella, P., Salas, M. and Mencía, M. (2016). Engineering permissive insertion sites in the bacteriophage Phi29 DNA-linked terminal protein. PLoS One, 11, 164901.
  • Salas, M. (2016). My scientific life. Bacteriophage 6, 1271250.
  • Suarez, C., Andrés, G., Kolovou, A., Hoppe, S., Salas, M. L., Walther, P. and Krijnse Locker, J. (2015). African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum. Cellular Microbiol. 17, 1683-1698.
  • Lacasta, A., Monteagudo, P. L., Jiménez-Marín, A., Accensi, F., Ballester, M., Argilaguet, J., Galindo, I., Segalés, J., Salas, M. L., Domínguez, J., Moreno, A., Garrido J. J. and Rodríguez, F. (2015) Live attenuated African swine fever viruses as ideal tools to dissect the mechanisms involved in viral pathogenesis and immune protection. Veterinary Research 46,135 DOI: 10.1186/s13567-015-0275-z.
  • Rodríguez, J. M., Moreno, L. T., Alejo, A., Lacasta, A., Rodríguez, F. and Salas, M. L. (2015) Genome sequence of African swine fever virus BA71, the virulent parental strain of the nonpathogenic and tissue-culture adapted BA71V. PLoS One Nov.30; 10 (11): e0142889. Doi: 10.1371/journal.pone.0142889. eCollection.
  • Hernaez, B., Guerra, M., Salas, M. L. and Andrés, G. (2016) The uncoating of African swine fever virus: a stepwise disassembly process that culminates in inner envelope fusion at multivesicular endosomes. PLoS Pathog 12 (4): e105595 doi:101371/journal.ppat 1005595.

 


 

Chapter book:

  • Salas, M. and de Vega, M. (2016). Protein-primed replication of bacteriophage ø29 DNA. In: Laurie S. Kaguni and Marcos Túlio Oliveira, editors, The Enzymes, Vol. 39, Burlington: Academic Press, pp 137-167.
  • de Vega, M., Lázaro, J.M. and Salas, M. (2016). Improvement of ø29 DNA polymerase amplification performance by fusión of DNA binding motifs. New Enzymes Useful in Rolling Circle Amplification (RCA). Springer, Demidov Ed. 10.1007/978-3-319-42226-8, pp 11-24.404.

 


 

Scientific Activities:

  • Co-dirigió la asignatura de Estabilidad de Genomas: Replicación, Reparación y Mutagénesis del Master Biología Molecular y Celular englobado en el Programa Oficial de Posgrado de Biociencias Moleculares de la Universidad Autónoma de Madrid (2015-2016 y 2016-2017).
  • Co-dirigió el Curso “Biomedicina y Biotecnología en la era genómica” de la Escuela de Biología Molecular “Eladio Viñuela”. Universidad Internacional Menéndez Pelayo (2015). Organizó la XIII y XIV Semana de la Ciencia del Ayuntamiento de Luarca. Asturias 2015 y 2016.
  • Co-dirigió el Curso “La superación de la crisis a través de la Ciencia” de la Escuela de Biología Molecular “Eladio Viñuela”. Universidad Internacional Menéndez Pelayo (2016).

 


 

Awards:

  • Denominación “Margarita Salas” al Bulevar del Parque Tecnológico de Andalucía. Málaga (2015–).
  • Medalla de Honor de la Real Academia Nacional de Medicina (2015).
  • Denominación “Margarita Salas” al IES Sevilla Este. Sevilla (2016–).
  • Medalla Echegaray de la Real Academia de Ciencias Exactas, Físicas y Naturales (2016).
  • Miembro del Consejo Rector de la Agencia Estatal de Investigación (2016–).

 


 

Doctoral Thesis:

  • Alicia del Prado Díaz (2015). Estudios estructurales y funcionales de la DNA polimerasa y la proteína terminal del bacteriófago phi29. Universidad Autónoma de Madrid. Directores: Margarita Salas y Miguel de Vega.
  • Isabel María Holguera López (2015). Estudio del dominio de unión a DNA de la proteína terminal del bacteriófago ø29 y su papel en la replicación del DNA viral. Universidad Autónoma de Madrid. Directores: Margarita Salas y Daniel Muñoz-Espín.
  • Pablo Gella Montero (2016). Optimización del sistema replicativo del bacteriófago phi29 para aplicaciones biotecnológicas. Universidad Autónoma de Madrid. Directores: Margarita Salas y Mario Mencía.

 


 

 ComFuturo-Vertical-Color-POS-Transp.png

 

The CSIC Foundation, through the program ComFuturo, is funding the contract of the researcher Modesto Redrejo Rodríguez for the project "New fusion DNA polymerases with biotechnological applications."

 


 

Currículum Vitae Margarita Salas

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