The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome

Molecular determinants of the DprA−RecA interaction for nucleation on ssDNA

International audienceNatural transformation is a major mechanism of horizontal gene transfer in bacteria that depends on DNA recombination. RecA is central to the homologous recombination pathway, catalyzing DNA strand invasion and homology search. DprA was shown to be a key binding partner of RecA acting as a specific mediator for its loading on the incoming exogenous ssDNA. Although the 3D structures of both RecA and DprA have been solved, the mechanisms underlying their cross-talk remained elusive. By combining molecular docking simulations and experimental validation, we identified a region on RecA, buried at its self-assembly interface and involving three basic residues that contact an acidic triad of DprA previously shown to be crucial for the interaction. At the core of these patches, DprA M238 and RecA F230 are involved in the interaction. The other DprA binding regions of RecA could involve the N-terminal ␣-helix and a DNA-binding region. Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA−RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA. Our model forms the basis for a mech-anistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA

Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes

Crystal Structure of the PP2A Phosphatase Activator: Implications for Its PP2A-Specific PPIase Activity

PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all α-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity

Evf, a virulence factor produced by the Drosophila pathogen Erwinia carotovora, is an S-palmitoylated protein with a new fold that binds to lipid vesicles

Erwinia carotovora are phytopathogenic Gram-negative bacteria of agronomic interest as these bacteria are responsible for fruit soft rot and use insects as dissemination vectors. The Erwinia carotovora carotovora strain 15 (Ecc15) is capable of persisting in the Drosophila gut by the sole action of one protein, Erwinia virulence factor (Evf). However, the precise function of Evf is elusive, and its sequence does not provide any indication as to its biochemical function. We have solved the 2.0-angstroms crystal structure of Evf and found a protein with a complex topology and a novel fold. The structure of Evf confirms that Evf is unlike any virulence factors known to date. Most remarkably, we identified palmitoic acid covalently bound to the totally conserved Cys209, which provides important clues as to the function of Evf. Mutation of the palmitoic binding cysteine leads to a loss of virulence, proving that palmitoylation is at the heart of Evf infectivity and may be a membrane anchoring signal. Fluorescence studies of the sole tryptophan residue (Trp94) demonstrated that Evf was indeed able to bind to model membranes containing negatively charged phospholipids and to promote their aggregation

Structure of the yeast Pml1 splicing factor and its integration into the RES complex

The RES complex was previously identified in yeast as a splicing factor affecting nuclear pre-mRNA retention. This complex was shown to contain three subunits, namely Snu17, Bud13 and Pml1, but its mode of action remains ill-defined. To obtain insights into its function, we have performed a structural investigation of this factor. Production of a short N-terminal truncation of residues that are apparently disordered allowed us to determine the X-ray crystallographic structure of Pml1. This demonstrated that it consists mainly of a FHA domain, a fold which has been shown to mediate interactions with phosphothreonine-containing peptides. Using a new sensitive assay based on alternative splice-site choice, we show, however, that mutation of the putative phosphothreonine-binding pocket of Pml1 does not affect pre-mRNA splicing. We have also investigated how Pml1 integrates into the RES complex. Production of recombinant complexes, combined with serial truncation and mutagenesis of their subunits, indicated that Pml1 binds to Snu17, which itself contacts Bud13. This analysis allowed us to demarcate the binding sites involved in the formation of this assembly. We propose a model of the organization of the RES complex based on these results, and discuss the functional consequences of this architecture

Etude structurale et fonctionnelle d'acteurs de la transformation génétique naturelle de Streptococcus pneumoniae

Streptococcus pneumoniae est la cause principale de pneumonies, otites, méningites et septicémies. La transformation génétique naturelle constitue l élément clé de son adaptation aux changements environnementaux. Elle s effectue par intégration d ADN d origine externe dans le chromosome de la bactérie, et a lieu pendant un état physiologique particulier appelé compétence.Mon travail de thèse a consisté à étudier les acteurs principaux de la régulation de la compétence (ComD, ComE) et les protéines impliquées dans la prise en charge, le traitement de l ADN transformant et la recombinaison (DprA, RecA). J ai notamment résolu la structure du facteur de transcription ComE par cristallographie aux rayons X, et réalisé une étude fonctionnelle de sa fixation sur un de ses promoteurs. Les résultats obtenus ont permis de proposer un mécanisme selon lequel la dimérisation induite par la phosphorylation de ComE, couplée à sa fixation sur la séquence promotrice d ADN, provoquerait une courbure de l ADN. Cette courbure permettrait la fixation de l ARN polymérase, activant ainsi la transcription des gènes nécessaires à la mise en place de la compétence.Streptococcus pneumoniae is the leading cause of community-acquired infections worldwide. The natural genetic transformation is the key to its adaptation to environmental changes. It takes place with the integration in its chromosome of exogenous DNA, during a physiological state called competence.During my thesis I have focused on the main actors of competence regulation (ComD, ComE) and on proteins involved in exogenous DNA processing and recombination (DprA, RecA). In particular, I have solved the structure of the transcriptional activator ComE by X-ray crystallography, and carried out a functional study of its binding to its promoter. The results obtained allowed us to propose a mechanism regarding the transcriptional activation by ComE of the genes necessary for the set up of the competence : the phosphorylation-induced dimerization, coupled to the binding of ComE to its DNA promoter, would curve the DNA and allow the binding of the RNA polymerase.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Etude structurale et fonctionnelle de DprA et de ses partenaires au cours de la transformation génétique naturelle

La transformation génétique naturelle est un mode de transfert horizontal de gènes chez les bactéries, qui contribue au maintien et à l'évolution de leurs génomes. C est un mécanisme clé pour l adaptation des bactéries, qui pourrait être responsable de la transmission des résistances aux antibiotiques observée en clinique chez certaines espèces pathogènes (S. pneumoniae, H. pylori, ). La transformation naturelle s effectue par l internalisation d ADN exogène à travers la membrane, puis par sa prise en charge jusqu à son intégration dans le chromosome bactérien par recombinaison homologue. Le processus de prise en charge fait intervenir la protéine DprA, très conservée dans le monde bactérien, impliquée dans la protection de l ADN entrant contre les nucléases, et dans le recrutement de la recombinase universelle RecA sur l ADNsb. DprA joue donc un rôle majeur et a récemment été décrite comme étant impliquée dans d autres aspects de la transformation génétique naturelle, comme la fermeture de la compétence via une interaction directe avec le régulateur de réponse ComE, ou la levée de la barrière du système de restriction-modification afin de faciliter la transformation. Chez H. pylori, DprA est en opéron avec DprB, suggérant l implication de ces 2 protéines dans une même voie et une interaction directe entre elles. DprA apparaît donc comme étant au cœur d un véritable réseau d interaction, protéique et nucléique.The natural genetic transformation is a mode of horizontal gene transfer that contributes to the maintenance and to the evolution of the genomes in bacteria. It is a key mechanism for their adaptation which could be responsible for the transmission of antibiotic resistances observed clinically for some pathogenic species (S. pneumoniae, H. pylori...). Natural transformation is performed by internalizing exogenous DNA followed by its processing and its integration into the bacterial chromosome by homologous recombination. The DNA processing involves the highly conserved DprA protein for the protection of the incoming DNA against nucleases and the recruitment of the universal recombinase RecA on ssDNA. DprA plays a key role and has recently been suggested to be involved in other aspects of the natural genetic transformation, such as the shut-off of the competence via a direct interaction with the response regulator ComE, or removal of the restriction-modification barrier system in order to facilitate the processing. In H. pylori, the dprA gene is in operon with dprB, whose function is unknown, suggesting their involvement in the same pathway and their likely direct interaction. DprA appears to be central in protein/nucleic acid interactions network.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

sHSPs under temperature and pressure: The opposite behaviour of lens alpha-crystallins and yeast HSP26

International audienceSmall angle X-ray scattering was used to follow the temperature and pressure induced structural transitions of polydisperse native calf lens alpha-crystallins and recombinant human alphaB-crystallins and of monodisperse yeast HSP26. The alpha-crystallins were known to increase in size with increasing temperature, whereas HSP26 partially dissociates into dimers. SAXS intensity curves demonstrated that the average 40-mer calf alpha-crystallin converted into 80-mer in a narrow temperature range, from 60 to 69 °C, whereas the average 30-mer alphaB-crystallin was continuously transformed into 60-mer at lower temperature, from 40 to 60 °C. These temperature-induced transitions were irreversible. Similar transitions, yet reversible, could be induced with pressure in the 100 to 300 MPa pressure range. Moreover, temperature and pressure could be combined to lower the transition temperatures. On the other hand, SAXS curves recorded during pressure scans from 0.1 to 200 MPa with monodisperse 24-mer HSP26 revealed dissociation of the 24-mer into dimers. This dissociation was complete and reversible. Whatever the sHSP, a decrease of partial specific volume was found to be associated with the pressure induced quaternary structure transitions, in agreement with the hypothesis that such transitions represent a first step on the protein denaturation pathway