Tesis doctoral de Candela Manfredi
In bacillus subtilis,with the exception of reca, recombination deficient mutants with increased sensitivities to dna-damaging agents have been isolated and classified within seven differen epistatic groups ( alpha, beta, gamma, delta, epsilon, zeta and eta). Mutants classified within the (recf, recl, reco and recr) epistatic group markedly affect the survival of cells exposed to dna-damaging agents. Sequencing data showed that recl16 strain is an unusual dominant-negative allele of the reco gene that contains a prematura stop codon at residue 37. This truncated peptide seems to alter recf protein localization in the cell. recr punctual mutations that have shown increased sensibility to mms would be located near the hole of the ring of recr tetramer, suggesting that they could be involved in reco and/or dna interaction. reca protein preferentially hydrolyzes datp than atp, and supports an efficient dna strand exchange in the presence of datp, even in the absence of the ssba protein, when compared to atp. Sub-saturating amounts of ssba, independent of the order of addition, inhibits atpase activity of reca and reduces the single-stranded (ss) dna-dependent datpase. This negative effect is fully overcome when a substoichiometric amount of reco is added, although, reco was unable to revert ssbspp1 inhibitory effects on reca datpase activity. ssba added prior to reca does not stimulate the datp-dependent dna strand exchange activity, however if added after reca it enhances the extent of strand exchange. Besides that, reca datp-dependent dna strand exchange activity is not stimulated by ssba. The addition of reco stimulates reca-mediated joint molecule formation in presence of datp, although it limits the accumulation of final recombination products. In presence of ssbspp1 reco has no effect, suggesting a reco-ssba specie- specific interaction. The reco promotes annealing of complementary ssdna in vitro.This activity is stimulated by ssba, but not by the heterologous single- stranded binding protein ssbspp1. The results suggest that once reco is bound to ssdna, it could enhance subsequent annealing of complementary región on the ssdnas by random interactions between several reco-adn complexes. Like rad52 in yeast, reco has ,a dual activity: it acts as a reca mediator enabling reca to ultilize ssba-coated ssdna and, by its annealing activity, is involved in the second end capture process. These activities are mediated by a specific interaction between reco and its cognate single-stranded binding protein. reca has a novel role in e. Coli behaviour connecting genome integrity and bacterial motility, it was observed that loss of reca specifically affeets swarming but not swimming motility. The results strongly suggest that the reca effect on swarrming motility does not require an extensive canonical reca nucleoprotein filament formation, because it is facilitated even by reca-gfp protein, which only have showed residual atpase activity and no nucleoprotein filament formation.
Datos académicos de la tesis doctoral «Estudio de los mediadores de reca en recombinación homóloga«
- Título de la tesis: Estudio de los mediadores de reca en recombinación homóloga
- Autor: Candela Manfredi
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 15/04/2009
Dirección y tribunal
- Director de la tesis
- Juan Carlos Alonso Navarro
- Tribunal
- Presidente del tribunal: andrés Aguilera lópez
- susana Campoy sánchez (vocal)
- José Antonio Tercero orduña (vocal)
- emilia Botello cambero (vocal)