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

Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction

International audienceThe histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-$T_{270}$ phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, $(R/K)R(I/A/V)$x$(L/P)$, the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-$F_{820}$ binds the same pocket of Asf1 as does histone $H4-F_{100}$. Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response

Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

International audienc

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase.

PNAS August 6, 2019 116 (32) 16018-16027; first published July 19, 2019Accurate chromosome segregation at mitosis and meiosis is crucial to prevent genome instability, birth defect, and cancer. Accordingly, separase, the protease that triggers chromosome distribution, is tightly regulated by a direct inhibitor, the securin. However, securin has not been identified, neither functionnally nor by sequence similarity, in other clades that fungi and animals. This raised doubts about the conservation of this mechanism in other branches of eukaryotes. Here, we identify and characterize the securin in plants. Despite extreme sequence divergence, the securin kept the same core function and is likely a universal regulator of cell division in eukaryotes

COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation in Arabidopsis*S⃞

Ubiquitination and proteasome-mediated degradation of proteins are crucial for eukaryotic physiology and development. The largest class of E3 ubiquitin ligases is made up of the cullin-RING ligases (CRLs), which themselves are positively regulated through conjugation of the ubiquitin-like peptide RUB/NEDD8 to cullins. RUB modification is antagonized by the COP9 signalosome (CSN), an evolutionarily conserved eight-subunit complex that is essential in most eukaryotes and cleaves RUB from cullins. The CSN behaves genetically as an activator of CRLs, although it abolishes CRL activity in vitro. This apparent paradox was recently reconciled in different organisms, as the CSN was shown to prevent autocatalytic degradation of several CRL substrate adaptors. We tested for such a mechanism in the model plant Arabidopsis by measuring the impact of a newly identified viable csn2 mutant on the activity and stability of SCFTIR1, a receptor to the phytohormone auxin and probably the best characterized plant CRL. Our analysis reveals that not only the F-box protein TIR1 but also relevant cullins are destabilized in csn2 and other Arabidopsis csn mutants. These results provide an explanation for the auxin resistance of csn mutants. We further observed in vivo a post-translational modification of TIR1 dependent on the proteasome inhibitor MG-132 and provide evidence for proteasome-mediated degradation of TIR1, CUL1, and ASK1 (Arabidopsis SKP1 homolog). These results are consistent with CSN-dependent protection of Arabidopsis CRLs from autocatalytic degradation, as observed in other eukaryotes, and provide evidence for antagonist roles of the CSN and 26S proteasome in modulating accumulation of the plant CRL SCFTIR1

Meet-U: Educating through research immersion.

We present a new educational initiative called Meet-U that aims to train students for collaborative work in computational biology and to bridge the gap between education and research. Meet-U mimics the setup of collaborative research projects and takes advantage of the most popular tools for collaborative work and of cloud computing. Students are grouped in teams of 4-5 people and have to realize a project from A to Z that answers a challenging question in biology. Meet-U promotes "coopetition," as the students collaborate within and across the teams and are also in competition with each other to develop the best final product. Meet-U fosters interactions between different actors of education and research through the organization of a meeting day, open to everyone, where the students present their work to a jury of researchers and jury members give research seminars. This very unique combination of education and research is strongly motivating for the students and provides a formidable opportunity for a scientific community to unite and increase its visibility. We report on our experience with Meet-U in two French universities with master's students in bioinformatics and modeling, with protein-protein docking as the subject of the course. Meet-U is easy to implement and can be straightforwardly transferred to other fields and/or universities. All the information and data are available at www.meet-u.org

Examples of strategies and results for the 2016–2017 edition.

<p>Left panel: Team B implemented an efficient sampling algorithm using a grid representation of the proteins to be docked and FFT. For the scoring, they used evolutionary information extracted from multiple sequence alignments of homologs of the two partners. Right panel: Team D used biological knowledge during the sampling step to filter out conformations early and drastically reduce the search space. The results obtained by the students (Teams B and D) on two complexes (barnase–barstar complex, Protein Data Bank [PDB] code: 1AY7, and an antibody–antigen complex, PDB code: 1JPS, respectively) are comparable to those obtained from state-of-the-art methods, namely ZDOCK [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref010" target="_blank">10</a>] and ATTRACT [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref011" target="_blank">11</a>]. ZDOCK relies on efficient sampling using FFT and on an optimized energy function [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref010" target="_blank">10</a>]. ATTRACT proceeds through minimization steps using an empirical, coarse-grained molecular mechanics potential [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref011" target="_blank">11</a>]. Candidate conformations for the complexes are represented as cartoons and superimposed onto the known crystallographic structures. The receptor is in black, the ligand from the candidate conformation is colored (in orange for Meet-U students, blue for ZDOCK, and purple for ATTRACT), and that from the crystallographic structure is in grey. With each candidate conformation are associated its rank, according to the scoring function of the method, and its deviation (in Å) from the crystallographic structure. FFT, Fast Fourier Transform; PDB, protein data bank.</p