Juan Carlos Alonso
Group Leader
Research summary
Nuestros objetivos son caracterizar la implicación de las funciones de reparación del ADN y la segregación cromosomal en la estabilidad del material genético en firmicutes. Para ello se utilizan dos modelos experimentales: Bacillus subtilis se usa para estudiar el papel de las proteínas de recombinación en la mecánica de la reparación de ADN y el plásmido pSM19035 para estudiar a la estabilidad segregacional.
Publications
Carrasco B, Escobedo S, Alonso JC, Suárez JE Modulation of Lactobacillus casei bacteriophage A2 lytic/lysogenic cycles by binding of Gp25 to the early lytic mRNA. Mol Microbiol 2016; 99: 328-337
Carrasco B, Serrano E, Sánchez H, Wyman C, Alonso JC. Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction. Nucleic Acids Res 2016 44: 2754-2768
Volante A, Carrasco B, Tabone M, Alonso JC. The interaction of omega with the RNA polymerase delta’ subunit functions as an activation to repression switch. Nucleic Acids Res 2015; 43: 9249-9261
Carrasco B, Yadav T, Serrano E, Alonso JC. Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair. Nucleic Acids Res 2015; 43: 5984-5997
Volante A, Alonso JC. Molecular anatomy of ParA-ParA and ParA-ParB interactions during plasmid partitioning. J Biol Chem 2015; 290: 18782-95
Our research focuses on the study of the molecular mechanisms that bacteria of the phylum Firmicutes use to secure the stability and horizontal transfer of genetic information, and to promote accurate segregation. Bacillus subtilis cells are chosen to study repair-by-recombination and plasmid segregation stability during vegetative growth as well as genetic recombination during natural competence. Using this model, we showed that the DNA damage response recruits different complex molecular machineries depending of the type of DNA damage, double-strand breaks (DSB) or single-strand gaps (SSG).
i) RecN, which is among the first responders to DSB, in concert with PNPase, promotes dynamic recruitment of DNA ends onto a repair centre. The AddAB complex or RecJ, in concert with the RecQ or RecS helicase and SsbA, creates a 3’-single-stranded (ss)DNA tailed duplex at the DNA breaks, and RecN recruits RecA mediators (SsbA, RecOR) and modulators (RecF, RecX, RecU, RecD) to initiate recombinational DNA repair (Fig. 1)
ii) distinct accessory proteins (DprA and SsbA and RecO(R) and SsbA) regulate recruitment and activation of RecA in the ATP-bound form to repair DSB and SSG (Fig. 1)
iii) CdaA and c-di-AMP are also involved in DSB repair, although their role is poorly understood
iv) in concert with RadA/Sms and c-di-AMP, DisA recognises recombination intermediates and modulates the repair of reversed forks by a poorly understood mechanism. Studying segregation in this bacterium, we showed that a) low copy number plasmids require homodimeric ParA-like (d2) and ParB-like (w2) parS regions for stable inheritance at cell division, b) the d2-ATP protein associates dynamically to the nucleoid, c) d2 bound to the nucleoid captures and tethers plasmid copies towards the nucleoid, d) at stoichiometric d2 and w2 concentrations, the latter facilitates ATP hydrolysis, creating a gradient of nucleoid-bound self-organizing d2 clouds, e) ATP hydrolysis leads to d2 disassembly from DNA, and the dynamic assembly/disassembly moves the plasmid molecule towards the cells poles to guarantee faithful segregation;, and f) if segregation fails, the toxin-antitoxin system halts proliferation of plasmid-free cells, with subsequent overgrowth of plasmid-bearing cells.
