CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOACaptura de pantalla 2022 09 14 a las 10.27.10    

Maintenance of bacterial genome stability

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 permanence of such damages could cause.

Our main objective is to get insights on the molecular mechanisms responsible for maintaining genome stability in bacteria, by functional analysis of the enzymatic features of purified repair proteins from model organisms as the gram positive bacterium Bacillus subtilis whose vegetative cells and spores have to handle DNA damage induced by extreme environmental conditions, and the gram negative Pseudomonas aeruginosa.

In this sense, during the 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, AP endonuclease, 3’-phosphatase and 3’-phosphodiesterase activities that share a common catalytic core at the C-terminal PHP domain, specifically present in the bacterial/archaeal subgroup of PolXs. Acting in concert with polymerization, those activities endow bacterial PolXs with the faculty to perform abasic site (AP) recognition, incision and further restoration (repair) of the original non-damaged nucleotide, as well as processing of the 3’-damaged ends that can arise after the exposition of the DNA to genotoxic agents.

Many bacterial members are provided with a nonhomologous end joining system (NHEJ) responsible for repairing double strand breaks (DSB), the most hazardous DNA lesions as they are lethal to dividing cells if they are not repaired in a timely fashion. Such a repair pathway is constituted by a Ku homodimer and a dedicated and multifunctional ATP-dependent Ligase (LigD). Previous biochemical analysis of bacterial LigD allowed the identification of polymerization, ligase and fosfoesterase activities. We have recently characterized the additional presence of an unexpected 5’-2-deoxyribose-5-phosphate (dRP) lyase activity in the ligase domain (LigDom) of the LigD from B. subtilis and P.aeruginosa. This activity coordinates with the polymerization and ligase activities to allow efficient repairing of an AP site-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. Therefore, LigD has in the same polypeptidic chain the three activities required in the last steps of BER, suggesting that its DNA repair role is not restricted to the NHEJ pathway, but expands beyond, being potentially active in additional repair pathways.

AP sites are the most common genomic DNA lesions frequently associated with DSBs. As their presence near a DSB end can pose a strong block to the final ligation, these lesions must be excised. Our last results show the presence of an additional AP-lyase activity in the PolDom of LigD that cleaves AP sites specifically when they are proximal to recessive 5’-ends and through the formation of a preternary precatalytic complex with Mn2+ ions and an incoming ribonucleotide complementary to the templating nucleotide opposite the AP site, guaranteeing the coupling of AP sites removal to the end-joining reaction by the bacterial LigD (see Figure 1).


Figure 1. Coupling of AP sites cleavage to the end-joining reaction by the B. subtilis LigD. After the breakage, the DNA end is threaded through the open ring-like structure of the Ku dimer (a). The location of the AP site proximal to the 5’-end could promote the partial melting of the 5’-end making the AP site accessible. After its recruitment by Ku, LigD would form a complex with the DNA, most probably implying the interaction of Lys331 with one of the phosphates of the phosphodiester bond between the AP site and the next 3’ nucleotide (b). The templating nucleotide opposite the AP site directs the binding of the complementary ribonucleotide, forming a Watson-Crick base pair at the polymerization site of the PolDom (c). Once the preternary-precatalytic complex is stabilized, the protein incises at the AP site, releasing the cleaved strand and giving rise to a new 5’-P end (d). PolDom mediates further synapsis between the 3’ overhanging strands from opposing breaks catalyzing the in trans addition of the nucleotide to the 3’-end of the incoming primer (e). Finally, the LigDom ligates both ends (f).


* For external calls please dial 34 91196 followed by the extension number
Last nameNameLaboratoryExt.*e-mailProfessional category
Alba FernándezLucía4094463lucia.alba(at)cbm.csic.esM1
Díaz ArcoSilvia 4094463silvia.diaz(at)cbm.csic.esM3 Predoc.formación
Prado DíazAlicia del4094463adelprado(at)cbm.csic.esE. Técnicos Especializados de Organismos Públicos de Investigación
Vega JoséMiguel de4094717mdevega(at)cbm.csic.esE. Investigadores Científicos de Organismos Públicos

Relevant publications:

  • Rodríguez, G., Martín, M.T. and de Vega, M. (2019) An array of basic residues is essential for the nucleolytic activity of the PHP domain of bacterial/archaeal PolX DNA polymerases. Sci. Rep. 9:9947
  • de Ory, A., Carabaña, C., and de Vega, M. (2019) Bacterial Ligase D preternary-precatalytic complex performs efficient abasic sites processing at double strand breaks during nonhomologous end joining. Nucleic Acids Res. 47(10), 5276-5292.
  • Fernández-García, J.L., de Ory, A., Brussaard, C.P.D. and 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. and 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. and 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. and 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., and 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.
  • Baños, B., Villar, L. Salas, M. and de Vega, M. (2010) Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites. Proc. Nat. Acad. Sci. USA 107(45): 19219-19224.
  • Baños, B., Lázaro, J.M., Villar, L., Salas, M. and de Vega, M. (2008) Editing of misaligned 3’-termini by an intrinsic 3’-5’ exonuclease activity residing in the PHP domain of a family X DNA polymerase. Nucleic Acids Res. 36(18): 5736-5749

Doctoral theses:

  • Irene Rodríguez García (2006). La DNA polimerasa del bacteriófago ø29: Análisis mutacional de la interacción con la proteína terminal. Base estructural de la procesividad y la capacidad de desplazamiento de banda. Universidad Autónoma de Madrid. Directores: Miguel de Vega & Margarita Salas
  • Patricia Pérez Arnaiz (2008). Relación estructura-función en la DNA polimerasa del bacteriófago ø29. Papel del dominio intermedio de la proteína terminal en el reconocimineto específico de la DNA polimerasa. Universidad Autónoma de Madrid. Director: Miguel de Vega
  • Elisa Longás Torné (2008). Caracterización funcional de las DNA polimerasas de los bacteriófagos Nf y GA-1. Estudio del mecanismo de iniciación en la replicación con proteína terminal. Universidad Autónoma de Madrid. Directores: Miguel de Vega & Margarita Salas
  • Benito Baños Piñero (2011). Caracterización funcional de la DNA polimerasa X de Bacillus subtilis. Universidad Autónoma de Madrid. Director: Miguel de Vega
  • Alicia del Prado Díaz (2015) Estudios estructurales y funcionales de la DNA polimerasa y la proteína terminal del bacteriófago ø29. Universidad Autónoma de Madrid. Directores: Miguel de Vega & Margarita Salas
  • Ana de Ory López (2016). Análisis bioquímico de las proteínas de reparación del DNA Ku y Ligasa D de Bacillus subtilis. Universidad Autónoma de Madrid. Director: Miguel de Vega
  • Mª Eugenia Santos del Río (2017). Papel del motivo LExE de la DNA polimerasas que inician con proteína terminal en la interacción con el nucleótido entrante. Estabilización de los sustratos en el centro activo de polimerización mediada por el subdominio TPR1. Universidad Autónoma de Madrid. Directores: Miguel de Vega & Margarita Salas


  • 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

NOTE! This site uses cookies and similar technologies.

If you not change browser settings, you agree to it. Learn more

I understand


What are cookies?

A cookie is a file that is downloaded to your computer when you access certain web pages. Cookies allow a web page, among other things, to store and retrieve information about the browsing habits of a user or their equipment and, depending on the information they contain and the way they use their equipment, they can be used to recognize the user.

Types of cookies

Classification of cookies is made according to a series of categories. However, it is necessary to take into account that the same cookie can be included in more than one category.

  1. Cookies according to the entity that manages them

    Depending on the entity that manages the computer or domain from which the cookies are sent and treat the data obtained, we can distinguish:

    • Own cookies: those that are sent to the user's terminal equipment from a computer or domain managed by the editor itself and from which the service requested by the user is provided.
    • Third party cookies: those that are sent to the user's terminal equipment from a computer or domain that is not managed by the publisher, but by another entity that processes the data obtained through the cookies. When cookies are installed from a computer or domain managed by the publisher itself, but the information collected through them is managed by a third party, they cannot be considered as own cookies.

  2. Cookies according to the period of time they remain activated

    Depending on the length of time that they remain activated in the terminal equipment, we can distinguish:

    • Session cookies: type of cookies designed to collect and store data while the user accesses a web page. They are usually used to store information that only is kept to provide the service requested by the user on a single occasion (e.g. a list of products purchased).
    • Persistent cookies: type of cookies in which the data is still stored in the terminal and can be accessed and processed during a period defined by the person responsible for the cookie, which can range from a few minutes to several years.

  3. Cookies according to their purpose

    Depending on the purpose for which the data obtained through cookies are processed, we can distinguish between:

    • Technical cookies: those that allow the user to navigate through a web page, platform or application and the use of different options or services that exist in it, such as controlling traffic and data communication, identifying the session, access to restricted access parts, remember the elements that make up an order, perform the purchase process of an order, make a registration or participation in an event, use security elements during navigation, store content for the broadcast videos or sound or share content through social networks.
    • Personalization cookies: those that allow the user to access the service with some predefined general characteristics based on a series of criteria in the user's terminal, such as the language, the type of browser through which the user accesses the service, the regional configuration from where you access the service, etc.
    • Analytical cookies: those that allow the person responsible for them to monitor and analyse the behaviour of the users of the websites to which they are linked. The information collected through this type of cookies is used in the measurement of the activity of the websites, applications or platforms, and for the elaboration of navigation profiles of the users of said sites, applications and platforms, in order to introduce improvements in the analysis of the data of use made by the users of the service.

Cookies used on our website

The CBMSO website uses Google Analytics. Google Analytics is a simple and easy to use tool that helps website owners to measure how users interact with the content of the site. You can consult more information about the cookies used by Google Analitycs in this link.

Acceptance of the Cookies Policy

The CBMSO assumes that you accept the use of cookies if you continue browsing, considering that it is a conscious and positive action from which the user's consent is inferred. In this regard, you are previously informed that such behaviour will be interpreted that you accept the installation and use of cookies.

Knowing this information, it is possible to carry out the following actions:

  • Accept cookies: if the user presses the acceptance button, this warning will not be displayed again when accessing any page of the portal.
  • Review the cookies policy: the user can access to this page in which the use of cookies is detailed, as well as links to modify the browser settings.

How to modify the configuration of cookies

Using your browser you can restrict, block or delete cookies from any web page. In each browser the process is different, here we show you links on this particular of the most used browsers: