Etude des mécanismes de recombinaison chez Arabidopsis thaliana (les protéines du groupe épistatique RAD52 et leur rôle dans la stabilité génomique)

CLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified from yeast (Rad55, Rad57 and Dmc1) to vertebrates (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 and Dmc1). These Rad51-like proteins are all members of the genetic recombination and DNA damage repair pathways. The sequenced genome of Arabidopsis thaliana encodes putative homologues of all six vertebrate Rad51-like proteins. We have identified and characterized an Arabidopsis mutant defective for one of these, AtXRCC3, the homologue of XRCC3. atxrcc3 plants are sterile, while they have normal vegetative development. Cytological observation shows that the atxrcc3 mutation does not affect homologous chromosome synapsis, but leads to chromosome fragmentation after pachytene, thus disrupting both male and female gametogenesis. This study shows an essential role for AtXrcc3 in meiosis in plants and possibly in other higher eukaryotes. Furthermore, atxrcc3 cells and plants are hypersensitive to DNA-damaging treatments, supporting the involvement of this Arabidopsis Rad51-like protein in recombinational repair

Perturbation of PALB2 function by the T413S mutation found in small cell lung cancer [version 2; referees: 3 approved]

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 corresponding ChAM mutations, and evaluated PALB2 chromatin association properties and the cellular resistance to camptothecin (CPT). Additionally, we examined the accumulation of γH2A.X and the RAD51 recombinase as readouts of DNA damage signalling and HDR, respectively. Results: We demonstrate that a small-cell lung cancer (SCLC)-associated T413S mutation in PALB2 impairs its chromatin association and confers reduced resistance to CPT, the only FDA-approved drug for relapsed SCLC. Unexpectedly, we found a less efficient γH2A.X nuclear foci formation in PALB2 T413S expressing cells, whereas a near-normal level of RAD51 nuclear foci was visible. Conclusions: These findings support the importance of PALB2 chromatin association in the suppression of tumours, including SCLC, an unusually aggressive type of cancer with poor prognosis. PALB2 T413S has little impact on RAD51 recruitment, likely due to its intact interaction with BRCA1 and BRCA2. However, this mutant shows inefficient DNA stress signalling. This finding sheds new light on the function of PALB2, playing a role in efficient DNA stress signalling through constitutive chromatin association

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis.

International audienceChromosomal 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 identified 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 find 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 efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants

KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity

The tumour suppressor PALB2 stimulates error-free repair of DNA breaks, whilst its steady-state chromatin association protects active genes from genotoxic stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/B), commonly known to promote transcriptional activation, acetylate the PALB2 chromatin association motif (ChAM), providing a dynamic regulatory mechanism for PALB2. ChAM acetylation within a cluster of seven lysine residues (7K), detected in the chromatin-enriched fraction in undamaged cells, enhanced its association with nucleosomes while decreasing its non-specific binding to naked DNA. DNA damage triggered a rapid deacetylation of ChAM and a concomitant increase in PALB2 mobility. Significantly, a 7K-null mutation, which hindered ChAM binding to both nucleosomes and DNA, conferred deficiency in DNA repair and hypersensitivity to the anti-cancer drug olaparib. Thus, our study reveals a unique mechanism mediated by KAT2A/B-dependent acetylation of a non-histone protein, which fine-tunes the DNA damage response and hence promotes genome stability.The tumour suppressor PALB2 stimulates error-free repair of DNA breaks, whilst its steady-state chromatin association protects active genes from genotoxic stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/B), commonly known to promote transcriptional activation, acetylate the PALB2 chromatin association motif (ChAM), providing a dynamic regulatory mechanism for PALB2. ChAM acetylation within a cluster of seven lysine residues (7K), detected in the chromatin-enriched fraction in undamaged cells, enhanced its association with nucleosomes while decreasing its non-specific binding to naked DNA. DNA damage triggered a rapid deacetylation of ChAM and a concomitant increase in PALB2 mobility. Significantly, a 7K-null mutation, which hindered ChAM binding to both nucleosomes and DNA, conferred deficiency in DNA repair and hypersensitivity to the anti-cancer drug olaparib. Thus, our study reveals a unique mechanism mediated by KAT2A/B-dependent acetylation of a non-histone protein, which fine-tunes the DNA damage response and hence promotes genome stability

KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity

The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.</p

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress

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. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes