Personne : Masson, Jean-Yves
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CHU de Québec-Université Laval. Centre de recherche
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- PublicationRestreintStructure and function of phage p2 ORF34p2, a new type of single-stranded DNA binding protein(Blackwell Scientific Publications, 2009-09-14) Scaltriti, Erika; Tremblay, Denise; Tegoni, Mariella; Masson, Jean-Yves; Rivetti, Claudio; Launay, Hélène; Moineau, Sylvain; Magadán, Alfonso H.; Ramoni, Roberto; Lichière, Julie; Campanacci, Valérie; Cambillau, Christian; Ortiz-Lombardía, MiguelLactococcus lactis, a Gram‐positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single‐stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single‐strand annealing protein. Two‐dimensional gel electrophoresis showed that ORF34p2 is expressed in large amounts during the early phases of phage infection, suggesting an important role in this process. Gel‐shift assays, surface plasmon resonance and atomic force microscopy demonstrated that ORF34p2 interacts with single‐strand DNA with nanomolar affinity. We also determined the crystal structure of ORF34p2 and showed that it bears a variation of the typical oligonucleotide/oligosaccharide binding‐fold of SSBs. Finally, we found that ORF34p2 is able to stimulate Escherichia coli RecA‐mediated homologous recombination. The specific structural and biochemical properties that distinguish ORF34p2 from other SSB proteins are discussed.
- PublicationAccès libreSynthesis, biological evaluation and structure-activity relationships of novel substituted N-phenyl ureidobenzenesulfonate derivatives blocking cell cycle progression in S-phase and inducing DNA double-strand breaks(American Chemical Society, 2012-07-13) Fortin, Sébastien; Masson, Jean-Yves; Vevey, Florence; Turcotte, Vanessa; Côté, Marie-France; Lacroix, Jacques M.; Coulombe, Yan.; C. Gaudreault, René.Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents.
- PublicationAccès libreInvestigation of the DNA damage response to SFOM-0046, a new small-molecule drug inducing DNA double-strand breaks(Nature Publishing Group, 2016-03-22) Fortin, Sébastien; Masson, Jean-Yves; Pauty, Joris; Velic, Denis; Côté, Marie-France; Rodrigue, Amélie2-Ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) is a novel anticancer agent that arrests cell cycle in S-phase and causes DNA replication stress leading to the phosphorylation of H2AX into γ-H2AX. First, using the M21, HT29, HT-1080 and HeLa cell lines, we confirmed that S-phase cell cycle arrest and γ-H2AX foci induction by SFOM-0046 is a general mechanism occurring in diverse cancer cell lines. In addition to γ-H2AX, SFOM-0046 activates preferentially ATR-Chk1 in M21 and HT29 cells while both ATR-Chk1 and ATM-Chk2 pathways are activated in HCT116 cells. Co-localization of SFOM-0046-induced 53BP1 foci with γ-H2AX foci validates that the DNA damage generated corresponds to double-strand-breaks (DSBs). Consistent with an S-phase arrest, SFOM-0046 treatment induces RAD51 foci formation but not DNA-PKcs foci, confirming that homologous recombination is the major DSB repair pathway targeted by the drug. Furthermore, using isogenic HCT116 p53+/+ and HCT116 p53−/− cells, we showed that p53 plays a key role in the survival mechanism to SFOM-0046. Finally, SFOM-0046 exhibits a dose-dependent antitumor activity on human fibrosarcoma HT-1080 tumours grafted onto chick chorioallantoic membranes without showing embryo toxicity even at high doses. Altogether, our results highlight SFOM-0046 as a very promising drug that induces a replication stress response.
- PublicationRestreintFunctional and structural basis for a bacteriophage homolog of human RAD52(ScienceDirect, 2008-08-05) Masson, Jean-Yves; Bransi, Ali; Moineau, Sylvain; Stasiak, Alicja Z.; Ploquin, Mickaël; Stasiak, Andrzej; Yu, Xiong; Egelman, Edward H.; Paquet, ÉricIn eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family  and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding  affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.
- PublicationAccès libreSynthesis and biological evaluation of novel N-phenyl ureidobenzenesulfonate derivatives as potential anticancer agents. Part 2. Modulation of the ring B(Elsevier, 2015-09-10) Fortin, Sébastien; Masson, Jean-Yves; Côté, Marie-France; Gagné-Boulet, Mathieu; Lacroix, Jacques M.; Moussa, HananeDNA double strand-breaks (DSBs) are the most deleterious lesions that can affect the genome of living beings and are lethal if not quickly and properly repaired. Recently, we discovered a new family of anticancer agents designated as N-phenyl ureidobenzenesulfonates (PUB-SOs) that are blocking the cells cycle progression in S-phase and inducing DNA DSBs. Previously, we have studied the effect of several modifications on the molecular scaffold of PUB-SOs on their cytocidal properties. However, the effect of the nature and the position of substituents on the aromatic ring B is still poorly studied. In this study, we report the preparation and the biological evaluation of 45 new PUB-SO derivatives substituted by alkyl, alkoxy, halogen and nitro groups at different positions on the aromatic ring B. All PUB-SOs were active in the submicromolar to low micromolar range (0.24–20 μM). The cell cycle progression analysis showed that PUB-SOs substituted at position 2 by alkyl, halogen or nitro groups or substituted at position 4 by a hydroxyl group arrest the cell cycle progression in S-phase. Interestingly, all others PUB-SOs substituted at positions 3 and 4 arrested the cell cycle in G2/M-phase. PUB-SOs arresting the cell cycle progression in S-phase also induced the phosphorylation of H2AX (γH2AX) which is indicating the generation of DNA DSBs. We evidenced that few modifications on the ring B of PUB-SOs scaffold lead to cytocidal derivatives arresting the cell cycle in S-phase and inducing γH2AX and DSBs. In addition, this study shows that these new anticancer agents are promising and could be used as alternative to circumvent some of the biopharmaceutical complications that might be encountered during the development of PUB-SOs.
- PublicationRestreintLactococcal phage p2 ORF35-Sak3 is an ATPase involved in DNA recombination and AbiK mechanism(Blackwell Scientific Publications, 2011-02-15) Scaltriti, Erika; Masson, Jean-Yves; Launay, Hélène; Moineau, Sylvain; Bron, Patrick; Genois, Marie-Michelle; Rivetti, Claudio; Ploquin, Mickaël; Grolli, Stefano; Campanacci, Valérie; Tegoni, Mariella; Cambillau, ChristianVirulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35p2, also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35p2 form a hexameric ringlike structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35p2 interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35p2 is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35p2 is a novel SSAP stimulating homologous recombination.
- PublicationRestreintDeciphering the function of lactococcal phage ul36 Sak domains(Academic Press, 2009-12-29) Scaltriti, Erika; Masson, Jean-Yves; Launay, Hélène; Moineau, Sylvain; Rivetti, Claudio; Ramoni, Roberto; Campanacci, Valérie; Tegoni, Mariella; Cambillau, ChristianVirulent phages are responsible for milk fermentation failures in the dairy industry, due to their ability to infect starter cultures containing strains of Lactococcus lactis. Single-strand annealing proteins (SSAPs) have been found in several lactococcal phages, among which Sak in the phage ul36. Sak has been recently shown to be a functional homolog of the human protein RAD52, involved in homologous recombination. A comparison between full-length Sak and its N- and C-terminal domains was carried out to elucidate functional characteristics of each domain. We performed HPLC-SEC, AFM and SPR experiments to evaluate oligomerization states and compare the affinities to DNA. We have shown that the N-terminal domain (1-171) is essential and sufficient for oligomerization and binding to DNA, while the C-terminal domain (172-252) does not bind DNA nor oligomerize. Modelisation of Sak N-terminal domain suggests that DNA may bind a positively charged crevice that runs external to the ring. Annealing and stimulation of RecA strand exchange indicate that only the N-terminal domain is capable of single-strand annealing and both domains do not stimulate the RecA strand exchange reaction. We propose that Sak N-terminus is involved in DNA binding and annealing while the C-terminus may serve to contact Sak partners.