Saturday, 23rd June 2018

Genome Dynamics and Function

        Bacterial DNA repair




Miguel de Vega




Research summary:

Maintenance of genome stability largely relies on faithful DNA replication. However, the continuous damage of the genomes by genotoxic agents has rendered necessary the emergence of repair mechanisms to prevent the deleterious effects that the permanence of such damages could cause.

Our main objective is to get insights on the molecular mechanisms responsible for maintaining genetic information in bacteria, by functional analysis of purified repair proteins from the bacterium Bacillus subtilis whose vegetative cells and spores have to deal with DNA damage induced by extreme environmental conditions.

In this sense, during last years we have been studying the catalytic functions of the B. subtilis DNA polymerase belonging to family X (PolXBs). We have shown that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3´-5´ exonuclease activity specialized in resecting unannealed 3´ termini in a gapped DNA substrate, being the first description of a family X DNA polymerase endowed with an editing activity. Biochemical analyses of PolXBs mutants have allowed us to demonstrate that such an activity resides at the C-terminal PHP domain, specifically present in the bacterial/archaeal subgroup of PolXs. Additionally, we have identified a novel AP-endonuclease activity, genetically linked to the 3´-5´ exonuclease one, that allows the enzyme to perform abasic site recognition, incision and further restoration (repair) of the original non-damaged nucleotide in a stand-alone AP-endonuclease independent way. Further biochemical analyses of site-directed mutants of PolXBs, together with its structural modeling using as template the crystallographic data from Thermus thermophilus PolX (ttPolX), have led us to propose a structural model that accounts for the coordination of the different catalytic activities of bacterial PolXs during repair of abasic sites and mispaired 3´ termini (see Figure 1).

In addition, we are studying how two pivotal proteins of the nonhomologous end joining DNA repair pathway, the B. subtilis Ligase D and Ku, perform the repair of double strand breaks, the most hazardous DNA lesions as they are lethal to dividing cells if they are not repaired in a timely fashion. 


Figure 1. A PolXBs binding model to coordinate the polymerization, AP-endonuclease and 3´-5´  exonuclease activities. (A) Model of the PHP motion. Catalytic residues of the PHP domain are represented as orange sticks. The 3´ terminal nucleotide of the upstream primer strand is represented as spheres at the polymerization active center. Curved arrow indicates the proposed movement of the PHP domain toward the upper surface (solvent accessible) of the PolX core (formed by the thumb, palm, fingers and 8-kDa subdomains). Such a rotation could be possible by virtue of the long linker that connects both polymerase portions, allowing the superficial PHP active site to reach and either remove a non-extendable 3´ terminus or catalyze hydrolysis at an AP site placed at the polymerization active site, as depicted in (B). Once the PHP domain goes back to the initial position, the entrance of an incoming nucleotide would promote further elongation of the resulting sanitized 3´ ends. Model for PolXBs was provided by the homology-modeling server SWISS-MODEL, using the crystallographic structure of the ternary complex of ttPolX as template (PDB code 3AUO).



Recent publications:

  • Fernández-García, JL, de Ory, A, Brussaard, CPD, de Vega, M. (2017) Phaeocystis globosa virus DNA polymerase: a "Swiss Army knife", multifunctional DNA polymerase-lyase-ligase for base excision repair. Sci. Rep. 7:6907
  • Zafra O, Pérez de Ayala L, de Vega M. (2017) The anti/syn conformation of 8´-oxo-7,8-dihydro-2´-deoxyguanosine is modulated by Bacillus subtilis PolX active site residues His255 and Asn263. Efficient processing of damaged 3´-ends. DNA Repair 52: 59-69
  • de Ory A, Nagler K, Carrasco B, Raguse M, Zafra O, Moeller R, de Vega M. (2016) Identification of a conserved 5´-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair. Nucleic Acids Res. 44(4): 1833-44
  • de Ory A, Zafra O, de Vega M. (2014) Efficient processing of abasic sites by bacterial nonhomologous end-joining ku proteins. Nucleic Acids Res. 42(21): 13082-95.
  • de Vega M. (2013) The minimal Bacillus subtilis nonhomologous end joining repair machinery. PLoS ONE 8(5): e64232.
  • Baños B, Villar L, Salas M, de Vega M. (2012) DNA stabilization at the Bacillus subtilis PolX core: a binding model to coordinate polymerase, AP-endonuclease and 3'-5' exonuclease activities. Nucleic Acids Res. 40(19):9750-62.


-Método para la replicación, amplificación o secuenciación de un ADN molde. Inventors: Margarita Salas Falgueras, Miguel de Vega José, José M. Lázaro Bolos, Luis Blanco Dávila, Mario Mencía Caballero. Owner:CSIC. Priority number: P200930412. Priority date: July 2, 2009. PCT/ES2010/070456 presented July 1, 2010. Licensed to XPol Biotech, S.L.

-Quimera de ADN polimerasa del fago ø29. Inventors: Margarita Salas Falgueras, Miguel de Vega José, José M. Lázaro Bolos, Luis Blanco Dávila, Mario Mencía Caballero. Owner: CSIC. Priority number: P200930413.  Priority date: July 2, 2009. PCT/ES2010/070454 presented on July 1, 2010. Licensed to XPol Biotech, S.L.

-Método de amplificación de ADN basado en los orígenes de replicación del bacteriófago phi29 y secuencias nucleotídicas asociadas.Inventors: Margarita Salas Falgueras, Mario Mencía Caballero,Miguel de Vega José, Pablo Gella Montero, José M. Lázaro Bolos. Owner: CSIC. Priority number: P201130288. Priority date: March 3, 2011.                   

Doctoral Theses

Irene Rodríguez García (2006). Bacteriophage ø29 DNA polymerase: Mutational analysis of the interaction with terminal protein. Structural base of the processivity and strand displacement capacity. Universidad Autónoma de Madrid.

Patricia Pérez Arnaiz (2008). Structure-function relationship in bacteriophage ø29 DNA polymerase. Role of the intermediate domain of the terminal protein in the specific recognition of the DNA polymerase. Universidad Autónoma de Madrid

Elisa Longás Torné (2008). Functional characterization of the DNA polymerases from bacteriophages Nf and GA-1. Estudy of the initiation mechanism in the replication with terminal protein. Universidad Autónoma de Madrid.

Benito Baños Piñero (2011). Functional characterization of bacillus subtlis DNA polymerase X. Universidad Autónoma de Madrid.

Alicia del Prado Díaz (2015). Structural and functional studies of the DNA polymerase and terminal protein of bacteriophage ø29. Universidad Autónoma de Madrid.

Ana de Ory López (2016). Biochemical analysis of the DNA repair proteins Ku and Ligase D from Bacillus subtilis. Universidad Autónoma de Madrid.