Biology subjects

Charbonnier, J.-B.

Publications and source records attributed to Charbonnier, J.-B..

7 recordsLinked to original sources

bioRxiv2026

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

molecular biology↗

bioRxiv2024

Restriction on Ku's Inward Translocation Caps Telomere Ends

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for preserving 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, e.g., Rap1 in Saccharomyces cerevisiae. Here, we reveal a direct role for Rap1 in the inhibition of Kus NHEJ function at telomeres. A single Rap1 molecule bound near a DNA end inhibits NHEJ in vivo without disrupting Ku presence. Consistent with this, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM and molecular modelling analysis of this complex shows that Rap1 obstructs Kus inward translocation on DNA - an essential step for NHEJ at broken ends. Nanopore sequencing of telomere fusions confirms the importance of this pathway in protecting native telomere ends. Collectively, our findings uncover a mechanism of telomere end protection mediated by restricting Kus inward translocation, a functional switch that prevents promiscuous NHEJ repair at telomeres.

molecular biology↗

bioRxiv2024

DNA polymerase Lambda is anchored within the NHEJ synaptic complex via Ku70/80

Non-homologous end joining (NHEJ) is the predominant pathway by which double-strand DNA breaks (DSBs) are repaired in mammals. To enable final break closure, various NHEJ end-processing factors respond to the chemistry of the damaged DNA ends. Amongst these factors is DNA polymerase lambda (Pol {lambda}), a member of the Pol X family. How members of the Pol X family engage with the NHEJ complex is unknown. Here, we present cryo-EM structures of Pol {lambda} in complex with the Ku70/80 DSB sensor whilst engaged with the DNA-PK holoenzyme in a long-range synaptic complex. These structures reveal a specific interaction site between Ku70/80 and the Pol {lambda} BRCT domain. The functionality of this interaction is assessed by generating point mutations on either side of the Pol {lambda} BRCT:Ku70/80 interface. Using these mutants in two orthogonal assays in cells (live protein recruitment at biphoton laser-damaged nuclear sites and transfection with an original gap-filling reporter plasmid) defines the molecular basis and essentiality of the BRCT domain for the recruitment and activity of the Pol {lambda} within the NHEJ complex. Ultimately, these data explain the role of this interaction in cell survival to DSBs. Finally, we propose a unified model for the interaction of the three Pol X family members bearing BRCT domains with the same site of Ku70/80.

biochemistry↗

bioRxiv2023

Ku-binding motifs in RAG2, XLF, PAXX and MRI support functional redundancy during V(D)J recombination

The interaction of several partners with Ku through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Here, we first established more specifically the function of KBMs in V(D)J recombination as the molecular basis of functional redundancy between XLF and the NHEJ proteins MRI and PAXX. Then, given the functional redundancy between RAG2 and XLF, we explored the hypothesis of a KBM-mediated interaction between RAG2 and Ku. Through sequence alignment and biophysical methods, we identified a KBM at the C-terminus of RAG2 (R2CT) that mediates its interaction with Ku both in vitro and in cellulo. Notably, we showed that R2CT/Ku interaction is independent of the RAG nuclease activity. Finally, we demonstrated that the respective KBMs of RAG2 and XLF support their functional redundancy for V(D)J recombination.

molecular biology↗

bioRxiv2023

The FIGNL1-FIRRM complex is required to complete meiotic recombination in the mouse and prevents massive DNA damage-independent RAD51 and DMC1 loading

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1s role during meiotic recombination in mammals remains unknown. Here, we deciphered the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1{Delta}N alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.

genetics↗

bioRxiv2020

Regulation of the MLH1-MLH3 endonuclease in meiosis

During prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables reciprocal exchange of DNA segments between homologous chromosomes, thus promoting genetic diversity in the progeny1. A successful completion of meiotic recombination requires nucleolytic processing of recombination intermediates. Genetic and cellular data implicated a pathway dependent on the putative MLH1-MLH3 (MutL{gamma}) nuclease in generating crossovers, but mechanisms that lead to its activation were unclear2-4. Here, we have biochemically reconstituted key elements of this pro-crossover pathway. First, we show that human MSH4-MSH5 (MutS{gamma}), which was known to support crossing over5-7, binds branched recombination intermediates and physically associates with MutL{gamma}. This helps stabilize the ensemble at joint molecule structures and adjacent dsDNA. Second, we show that MutS{gamma} directly stimulates DNA cleavage by the MutL{gamma} endonuclease, which demonstrates a novel and unexpected function for MutS{gamma} in triggering crossing-over. Third, we find that MutL{gamma} activity is further stimulated by EXO1, but only when MutS{gamma} is present. Fourth, we also identify the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) as additional components of the nuclease ensemble, and show that S. cerevisiae strains expressing PIP box-mutated MutL{gamma} present striking defects in forming crossovers. Finally, we show that the MutL{gamma}-MutS{gamma}-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, the multilayered nuclease ensemble likely processes meiotic recombination intermediates by nicking dsDNA adjacent to junction points8. Since DNA nicking by MutL{gamma} is dependent on its co-factors, the asymmetric distribution of MutS{gamma} and RFC/PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This unique mode of MutL{gamma} nuclease activation might explain crossover-specific processing of Holliday junctions within the meiotic chromosomal context3, 9.

biochemistry↗

bioRxiv2019

Mechanism of in vivo activation of the MutLγ-Exo1 complex for meiotic crossover formation

Crossovers generated during the repair of programmed double-strand breaks (DSBs) during homologous recombination are essential for fertility to allow accurate homolog segregation during the first meiotic division. Most crossovers arise through the cleavage of recombination intermediates by the Mlh1-Mlh3 (MutL{gamma}) endonuclease and an elusive non-catalytic function of Exo1, and require the Polo kinase Cdc5. Here we show in budding yeast that MutL{gamma} forms a constitutive complex with Exo1, and in meiotic cells transiently contacts the Msh4-Msh5 (MutS{gamma}) heterodimer, also required for crossover formation. We further show that MutL{gamma}-Exo1 associates with recombination intermediates once they are committed to the crossover repair pathway, and then Exo1 recruits Cdc5 through a direct interaction that is required for activating MutL{gamma} and crossover formation. Exo1 therefore serves as a non-catalytic matchmaker between Cdc5 and MutL{gamma}. We finally show that in vivo, MutL{gamma} associates with the vast majority of DSB hotspots, but at a lower frequency near centromeres, consistent with a strategy to reduce at-risk crossover events in these regions. Our data highlight the tight temporal and spatial control of the activity of a constitutive, potentially harmful, nuclease.

molecular biology↗