Didier Busso

Separase plays central roles in chromosome separation during mitosis and in the centrosome duplication cycle. Tight control of separase activity is required to prevent unscheduled resolution of sister chromatid cohesion and centrosome aberrations, thereby maintaining genome stability. In mammals, despite their disassembly during early mitosis, some nuclear envelope components have mitotic roles, but the links with separase activity remain unexplored. Here, we uncover a novel mechanism of separase regulation involving lamin B1, a key nuclear envelope factor. We show that separase and lamin B1 associate preferentially during early mitotic stages. Importantly, lamin B1 depletion leads to an increase in separase recruitment to chromosomes together with premature chromatid separation, a phenotype reminiscent of separase overexpression. Conversely, similar to separase depletion, lamin B1 overexpression induces diplochromosome formation, together with centrosome amplification, phenotypes consistent with impaired sister chromatid separation. Importantly, increasing separase levels prevents lamin B1-induced centrosome aberrations, suggesting a separase defect at their origin. Indeed, we show that overexpression of lamin B1 leads to a decrease in the recruitment of separase to chromosomes and a delay in its activity. Taken together, this study unveils a novel mechanism of separase regulation involving the nuclear envelope factor lamin B1 that is critical for genome integrity.

Neural progenitor cell (NPC) migration is essential for proper cortical development, and its disruption contributes to severe brain malformations such as l

Acute megakaryoblastic leukemia (AMKL) is a rare and aggressive pediatric acute myeloid leukemia subtype, frequently driven by the ETO2::GLIS2 (EG) gene fusion in de novo AMKL patients. The EG fusion confers poor prognosis and high relapse risk. In a previous study, we demonstrated that human fetal hematopoietic cells are intrinsically permissive to EG-driven transformation and that microenvironmental cues critically enhance leukemogenesis in less permissive human cord blood (CB) cells. However, most pediatric AML models are established in adult recipient mice, potentially overlooking developmental niche-specific effects. Here, we investigated whether mouse neonates provide a more permissive environment for EG-driven leukemogenesis from human CB hematopoietic stem and progenitor cells (HSPCs). Comparison of xenotransplants of EG-transduced CB CD34 ⁺ cells into sublethally irradiated NSG neonate or adult mice show that neonatal recipients are more supportive of EG-driven leukemia when cell doses are adjusted for recipient size. Neonates exhibited a higher frequency of leukemia-initiating cells (LIC), enhanced expansion of EG/GFP ⁺ leukemic cells, and increased disease penetrance compared with adults at similar dose/size ratio, despite similar overall human hematopoietic engraftment. Leukemic cells arising in neonates and adults displayed comparable phenotypes and secondary transplantation capacities, indicating functional equivalence. Both CD34 ⁺ CD38 ^- and CD34 ⁺ CD38 ⁺ CB subpopulations were sensitive to EG-driven transformation, with neonates consistently showing higher LIC frequencies than adults. Together, our findings demonstrate that the developmental stage of the host microenvironment is a critical extrinsic determinant of EG-mediated leukemogenesis. Neonatal recipients constitute a physiologically relevant and highly sensitive model for studying pediatric AMKL initiation with low cell inputs, enabling refined investigation of microenvironmental signals that promote leukemia development and may reveal novel therapeutic vulnerabilities

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, Rap1 in Saccharomyces cerevisiae. Here, we find that Rap1 directly inhibits Ku’s NHEJ function at telomeres. A single Rap1 molecule near a double-stand break suppresses NHEJ without displacing Ku in cells. Furthermore, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM shows Rap1 blocks Ku’s inward translocation on DNA – an essential step for NHEJ at DSBs. Nanopore sequencing of telomere fusions confirms this mechanism protects native telomere ends. These findings uncover a telomere protection mechanism where Rap1 restricts Ku’s inward translocation. This switches Ku from a repair-promoting to a protective role preventing NHEJ at telomeres.

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks and the restart of stalled replication forks. A critical step in HR is the formation of Rad51 nucleofilaments, which perform homology search and strand invasion of a homologous DNA sequence required for repair synthesis. In the yeast Saccharomyces cerevisiae, Rad52 facilitates Rad51 nucleofilament formation by mediating Rad51 loading onto ssDNA and counteracting Rad51 filament dissociation by the DNA translocase Srs2. The molecular basis of these two Rad52 functions remains unclear. Our integrative structural analyses of the Rad51-Rad52 interaction, combining NMR, SAXS, and modeling, reveal that an 85-residue segment of Rad52, conserved in fungi, folds upon binding to a broad surface of a Rad51 monomer. Notably, it includes an FxxA motif conserved in the BRC repeats of BRCA2 and at the Rad51-Rad51 interface. This binding mode was validated through an extensive set of mutations. Using in vivo assays and a functional fluorescent GFP-Rad51 fusion protein, we demonstrated that this entire segment is critical for Rad51 filament formation. These findings highlight how Rad52 functions as an assembly chaperone by preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and counteracting the effects of Srs2 on destabilizing Rad51 filaments.

Separase plays a central role in chromosome separation during mitosis and in centrosome cycle. Tight control of separase activity is required to prevent unscheduled resolution of sister chromatid cohesion and centrosome aberrations, thereby preserving genome stability. In mammals, despite their disassembly in early mitosis, some nuclear envelope components possess mitotic roles, but links with separase activity remain unexplored. Here, we uncover a new mechanism of separase regulation involving lamin B1, a key nuclear envelope factor. We show that separase and lamin B1 associate preferentially during early stages of mitosis. Importantly, lamin B1 depletion leads to an increase in separase recruitment on chromosomes together with premature chromatid separation, a phenotype reminiscent of separase overexpression. Conversely, similar to separase depletion, lamin B1 overexpression induces formation of diplochromosomes- resulting from chromatid separation failure-, in association with centrosome amplification. Importantly, increasing separase level prevents lamin B1-induced centrosome aberrations, suggesting a separase defect at their origin. Indeed, we show that overexpression of lamin B1 leads to a decrease in the recruitment of separase to the chromosome and a delay in its activity. Taken together, this study unveils a novel mechanism of separase regulation involving the nuclear envelope factor lamin B1, that is crucial for genome integrity maintenance.

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.

Background: Mirror movements are involuntary movements of one hand that mirror intentional movements of the other hand. Congenital mirror movements (CMM) is a rare genetic disorder with autosomal dominant inheritance, in which mirror movements are the main neurological manifestation. CMM is associated with an abnormal decussation of the corticospinal tract, a major motor tract for voluntary movements. RAD51 is known to play a key role in homologous recombination with a critical function in DNA repair. WhileRAD51haploinsufficiency was first proposed to explain CMM, other mechanisms could be involved.Methods: We performed Sanger sequencing ofRAD51in five newly identified CMM families to identify new pathogenic variants. We further investigated the expression of wild-type and mutant RAD51 in the patients’ lymphoblasts at mRNA and protein levels. We then characterised the functions of RAD51 altered by non-truncating variants using biochemical approaches.Results: The level of wild-type RAD51 protein was lower in the cells of all patients with CMM compared with their non-carrier relatives. The reduction was less pronounced in asymptomatic carriers.In vitro, mutant RAD51 proteins showed loss-of-function for polymerisation, DNA binding and strand exchange activity.Conclusion: Our study demonstrates thatRAD51haploinsufficiency, including loss-of-function of non-truncating variants, results in CMM. The incomplete penetrance likely results from post-transcriptional compensation. Changes in RAD51 levels and/or polymerisation properties could influence guidance of the corticospinal axons during development. Our findings open up new perspectives to understand the role of RAD51 in neurodevelopment.

Lamin B1 controls 53BP1 recruitment to DNA damage.

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how theSaccharomyces cerevisiaeORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (BlocksAddition ofTelomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5’ end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5’ resection.