Monday, 21st April 2014

Genome Dynamics and Function

Protein-primed replication of bacteriophage ø29 DNA





Margarita Salas











Research summary:


Modeling of the chimerical DNA polymerase. The figure represents the structural model of a (HhH)2 domain (colored in cyan) joint through a linker peptide (in dark blue) to the C-terminus of f29 DNA polymerase (colored in violet). 



Nuclear localization of bacteriophage terminal proteins (TP).


We have continued with the study of the structural and functional bases of the mechanism of ø29 DNA replication initiated by terminal protein (TP) priming. We have characterized the subdomains in the ø29 DNA polymerase and TP that determine the specificity in the initiation of replication and we have shown that the TP priming domain specifies the internal template nucleotide to initiate ø29 DNA replication. We have shown the involvement of the TPR2 subdomain in the high processivity and strand displacement capacity of the polymerase. In addition, movement of this subdomain is required for the activities of the ø29 DNA polymerase in TP-DNA replication and in circular DNA replication. In collaboration with Dr. Tom Steitz (Yale University) the 3D structure of ø29 DNA polymerase alone or in complex with TP as well as with DNA in the absence or presence of nucleotide have been determined, and the mechanism of translocation has been established. We have shown that the ø29 TP directs early organization of ø29 DNA replication at the bacterial nucleoid recruiting the phage DNA polymerase. Later on, both proteins, as well as the ø29 protein p16.7, form helical structures at the periphery of the infected cells dependent on the three MreB actin-like cytoskeleton proteins. These MreB proteins are required for the efficient replication of ø29 DNA. On the other hand, the ø29 histone-like protein p6 localizes in a peripheral helix-like configuration at early infection stages. Later, at middle infection times, protein p6 is recruited to the bacterial nucleoid. This migrating process was shown to depend on the synthesis of the viral DNA.

We have characterized the ø29 protein p56 as an inhibitor of the bacterial uracil-DNA glycosylase (UDG) by inhibiting its binding to DNA, thus preventing the ø29 DNA replication impairment caused by the uracil excision activity of UDG. In collaboration with Dr. Juan Luis Asensio (Institute of Organic Chemistry, CSIC, Madrid) and Dr. Beatriz González (Institute Rocasolano, CSIC, Madrid), the 3D structure of protein p56 and the UDG-p56 complex have been determined.

On the other hand, we have determined that the TPs of five different phages, among them ø29, have nuclear localization signals that may act as a putative horizontal gene transfer mechanism.

From the biotechnological point of view we have improved the amplification performance of ø29 DNA polymerase by fusion of DNA binding motifs. In addition, by changing the amplification conditions, we have reduced 103 to 104-fold the amount of DNA that can be amplified. We have also developed a DNA amplification system primed by the ø29 TP based on the minimal replication origins of ø29 DNA. This method opens up possibilities in relation to amplification of DNA and the generation of hybrid protein-DNA molecules.



G. Serrano-Heras, A. Bravo and M. Salas. (2008). Phage ø29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase. Proc.Natl.Acad. Sci.USA. 105, 19044-19049.

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.



Curriculum Vitae Margarita Salas