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).
| Last name | Name | Laboratory | Ext.* | Professional category | |
|---|---|---|---|---|---|
| Alba Fernández | Lucía | 405 | 4463 | lucia.alba(at)cbm.csic.es | M1 |
| Díaz Arco | Silvia | 405 | 4463 | silvia.diaz(at)cbm.csic.es | M3 Predoc.formación |
| Prado Díaz | Alicia del | 405 | 4463 | adelprado(at)cbm.csic.es | E. Técnicos Especializados de Organismos Públicos de Investigación |
| Vega José | Miguel de | 405 | 4717 | mdevega(at)cbm.csic.es | E. 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
Patents:
- 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
