Jean-Yves Bleuyard - Academia.edu (original) (raw)

Papers by Jean-Yves Bleuyard

Rad51 point mutations were introduced using QuikChange Site-Directed Mutagenesis Kit (Stratagene)... more Rad51 point mutations were introduced using QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s protocol, and were confirmed by DNA sequencing. For mammalian expression, human Rad51 was cloned into pcDNA5/FRT vector (Invitrogen) with or without FLAG epitope at N-terminus. Rad51 NTD (1-86) variants were PCR cloned into pET11d (Stratagene) or pGEX-4T-3 (GE Healthcare), and the recombinant proteins were purified following

Les voies de recombinaison homologue permettent des echanges d'information genetique entre de... more Les voies de recombinaison homologue permettent des echanges d'information genetique entre des molecules d'ADN presentant une homologie de sequence. Outre leur role dans l'augmentation de la diversite genetique, ces voies contribuent a la reparation des lesions de l'ADN, ainsi qu'a la segregation des chromosomes homologues en cours de meiose. Chez les eucaryotes la recombinaison homologue depend principalement des proteines du groupe epistatique RAD52. En l'etat actuel, peu de proteines de ce groupe ont ete identifiees et caracterisees chez les plantes. Chez les vertebres, les proteines Rad51B, Rad51C, Xrcc2, Xrcc3 (toutes membres du groupe epistatique RAD52) sont necessaires a la reparation des lesions de l'ADN et aux evenements de recombinaison mitotique. Cependant, un phenotype de letalite embryonnaire a considerablement limite l'etude de leur role dans les processus de la meiose. Dans le cadre de l'etude des mecanismes de recombinaison chez la...

Wellcome Open Research

Background: Germline mutations in the PALB2 gene are associated with the genetic disorder Fanconi... more Background: Germline mutations in the PALB2 gene are associated with the genetic disorder Fanconi anaemia and increased predisposition to cancer. Disease-associated variants are mainly protein-truncating mutations, whereas a few missense substitutions are reported to perturb its interaction with breast cancer susceptibility proteins BRCA1 and BRCA2, which play essential roles in homology-directed repair (HDR). More recently, PALB2 was shown to associate with active genes independently of BRCA1, and through this mechanism, safeguards these regions from DNA replicative stresses. However, it is unknown whether PALB2 tumour suppressor function requires its chromatin association. Methods: Mining the public database of cancer mutations, we identified four potentially deleterious cancer-associated missense mutations within the PALB2 chromatin association motif (ChAM). To assess the impact of these mutations on PALB2 function, we generated cell lines expressing PALB2 variants harbouring cor...

The tumour suppressor PALB2 stimulates error-free DNA repair by homologous recombination (HR). It... more The tumour suppressor PALB2 stimulates error-free DNA repair by homologous recombination (HR). It also associates with actively transcribed chromatin and protects these regions from DNA damage. However, the mechanism by which PALB2 switches between these functions remains enigmatic. Here, we show that the PALB2 chromatin association motif (ChAM), which directly binds to nucleosomes, is targeted by acetylation at a cluster of seven lysine residues (the '7K-patch'). ChAM acetylation within the 7K-patch enhances its nucleosome binding, while limiting its non-specific DNA binding. Importantly, ChAM acetylation is rapidly removed upon DNA damage, resulting in reduced PALB2 chromatin association and increased mobility within the nucleus. The 7K-null mutation confers HR deficiency, including increased sensitivity to the anti-cancer drug Olaparib. Thus, our findings reveal a unique mechanism mediated by acetylation of non-histone protein, which regulates the chromatin interaction of...

Proceedings of the National Academy of Sciences of the United States of America, Jul 18, 2017

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome int... more The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment....

Cell reports, Jan 12, 2014

Numerous human genome instability syndromes, including cancer, are closely associated with events... more Numerous human genome instability syndromes, including cancer, are closely associated with events arising from malfunction of the essential recombinase Rad51. However, little is known about how Rad51 is dynamically regulated in human cells. Here, we show that the breast cancer susceptibility protein BRCA2, a key Rad51 binding partner, coordinates the activity of the central cell-cycle drivers CDKs and Plk1 to promote Rad51-mediated genome stability control. The soluble nuclear fraction of BRCA2 binds Plk1 directly in a cell-cycle- and CDK-dependent manner and acts as a molecular platform to facilitate Plk1-mediated Rad51 phosphorylation. This phosphorylation is important for enhancing the association of Rad51 with stressed replication forks, which in turn protects the genomic integrity of proliferating human cells. This study reveals an elaborate but highly organized molecular interplay between Rad51 regulators and has significant implications for understanding tumorigenesis and the...

The Plant Journal, 2004

In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the... more In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51-dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross-linking agent Mitomycin C, but not to gamma-irradiation. Moreover, the atrad51c-1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.

The Plant Journal, 2003

Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (N... more Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identi®ed a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We ®nd that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the ef®ciency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants.

Plant Molecular Biology, 2004

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation... more Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed doublestrand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an Arabidopsis Rad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un-or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.

EMBO reports, 2011

The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the re... more The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the repair of DNA damage by homologous recombination. Here, we report that chromatin-association motif (ChAM), an evolutionarily conserved motif in PALB2, is necessary and sufficient to mediate its chromatin association in both unperturbed and damaged cells. ChAM is distinct from the previously described PALB2 DNA-binding regions. Deletion of ChAM decreases PALB2 and Rad51 accumulation at DNA damage sites and confers cellular hypersensitivity to the genotoxic drug mitomycin C. These results suggest that PALB2 chromatin association via ChAM facilitates PALB2 function in the cellular resistance to DNA damage.

DNA Repair, 2006

Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA ... more Living cells suffer numerous and varied alterations of their genetic material. Of these, the DNA double-strand break (DSB) is both particularly threatening and common. Double-strand breaks arise from exposure to DNA damaging agents, but also from cell metabolism-in a fortuitous manner during DNA replication or repair of other kinds of lesions and in a programmed manner, for example during meiosis or V(D)J gene rearrangement. Cells possess several overlapping repair pathways to deal with these breaks, generally designated as genetic recombination. Genetic and biochemical studies have provided considerable amounts of data about the proteins involved in recombination processes and their functions within these processes. Although they have long played a key role in building understanding of genetics, relatively little is known at the molecular level of the genetic recombination processes in plants. The use of reverse genetic approaches and the public availability of sequence tagged mutants in Arabidopsis thaliana have led to increasingly rapid progress in this field over recent years. The rapid progress of studies of recombination in plants is obviously not limited to the DSB repair machinery as such and we ask readers to understand that in order to maintain the focus and to rest within a reasonable length, we present only limited discussion of the exciting advances in the of plant meiosis field, which require a full review in their own right [1,2]. We thus present here an update on recent advances in understanding of the DSB repair machinery of plants, focussing on Arabidopsis and making a particular effort to place these in the context of more general of understanding of these processes.

Chromosoma, 2004

The Rad50, Mre11 and Xrs2/Nbs1 proteins, which form the highly conserved MRX complex, perform a w... more The Rad50, Mre11 and Xrs2/Nbs1 proteins, which form the highly conserved MRX complex, perform a wide range of functions concerning the maintenance and function of DNA in eukaryotes. These include recombination, DNA repair, replication, telomere homeostasis and meiosis. Notwithstanding the attention paid to this complex, the inviability of vertebrate rad50 and mre11 mutants has led to a relative lack of information concerning the role of these proteins in meiosis in higher eukaryotes. We have previously reported that Arabidopsis atrad50 mutant plants are viable and that atrad50 mutant plants are sterile. The present study reports an analysis of the causes of this sterility and the implication of the AtRad50 protein in meiosis. Both male and female gametogenesis are defective in the Arabidopsis atrad50 mutant and cytological observation of male meiosis indicates that in the absence of the AtRad50 protein, homologous chromosomes are unable to synapse. Finally, the atrad50 mutation leads to the destruction of chromosomes during meiosis. These phenotypes support a role for the Arabidopsis MRX complex in early stages of meiotic recombination.