Friday, 31st July 2015

Genome Dynamics and Function

     Replication of Bacteriophage ø29 DNA

 

 

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Margarita Salas

 

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Research summary:

We have continued with the study of the mechanism of ø29 DNA replication initiated by TP-priming. We have optimized the ø29 replication origins for TP-primed initiation of replication. Ø29 DNA polymerase Lys529 is involved in the stabilization of the primer-terminus in the polymerization active site. This lysine and Glu233 of the TP establish contacts for the initiation of TP-DNA replication. The LExE motif, conserved in eukaryotic-type DNA polymerases, is involved in the interaction with the incoming nucleotide. Residues Tyr226 and Tyr390 in the ø29 DNA polymerase active site are involved in the mechanism of translocation and in dNTP and pyrophosphate binding. In the ø29 TP there is a correlation between the capacity of DNA binding and nucleoid localization. On the other hand, the nuclear and nucleoid localization of the TPs of ø29 and other bacteriophages are independently conserved functions. The ø29 protein p1 associates with the FtsZ ring of the Bacillus subtilis divisome producing an increase in he cellular length and in the accumulation of the viral DNA. In collaboration with Dr. Beatriz González we have obtained the three-dimensional structure of the B. subtilis uracil-DNA glycosilase (UDG) free and in complex with the ø29 protein p56 and we have determined key amino acids for the UDG inhibition by p56.


 Fig1 300  

Three-dimensional structure of the UDG-p56 complex

   
Fig2 300  
Colocalization of ø29 protein p1 and B. subtilis protein FtsZ

 

 

 

 

 

 

 

Relevant publications:

  • M. de Vega, J.M. Lázaro, M. Mencía, L. Blanco and M. Salas. (2010). Improvement of ø29 DNA polymerase amplification performance by fusion of DNA binding motifs. Proc.Natl.Acad.Sci.USA. 107, 16506-16511.
  • D. Muñóz-Espín, I. Holguera, D. Ballesteros-Plaza, R. Carballido-López and M. Salas (2010). Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid. Proc.Natl.Acad.Sci.USA. 107, 16548-16553.
  • M. Mencía, P. Gella, A. Camacho, M. de Vega and M. Salas (2011). Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage ø29. Proc.Natl.Acad.Sci.USA 108, 18655-18660.
  • M. Redrejo-Rodríguez, D. Muñoz-Espín, I. Holguera, M. Mencía and M. Salas (2012). Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages. Proc.Natl.Acad.Sci.USA 109, 18482-18487.
  • I.Holguera, D. Muñoz-Espín and M. Salas (2015). Dissecting the role of DNA-binding residues of the ø29 terminal protein in viral DNA replication. Nucleic. Acids Res. 43, 2790-2801

 

Doctoral Theses:

David Ballesteros Plaza (2014) Estudio funcional de las proteínas p1 y p17 del bacteriófago ø29. Universidad Autónoma de Madrid. Directores: Margarita Salas Falgueras y Daniel Muñoz Espín.


 

Margarita Salas CV