Biology subjects

Le Cam, E.

Publications and source records attributed to Le Cam, E..

8 recordsLinked to original sources

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

Imperfect hairpins formed by CTG trinucleotide repeats are heteroduplex DNA molecules in living cells

There are over fifty microsatellite expansion disorders, often neurodegenerative pathologies, due to large expansions of tandem repeat sequences. These microsatellites vary in motif composition, size and copy number in the genome. The ability of these repeats to form secondary structures in vitro is strongly suspected to trigger the expansion process and therefore, the pathology. However, there is no formal proof that similar structures are formed in vivo, in living and replicating cells. We are focused on the CTG trinucleotide repeat whose expansion is responsible for myotonic dystrophy type 1 (DM1 or Steinert disease), affecting 1 in 8,000 people worldwide. A series of llama nanobodies (VHH) were selected to bind to the secondary structure formed by a synthetic imperfect CTG hairpin. Yeast one-hybrid was used to identify VHH that were able to activate a reporter gene in vivo . One of them was shown to be very specific since it binds a CTG hairpin but not a CAG hairpin or a GC-rich sequence. This nanobody also bind to long CTG trinucleotide repeats carried by plasmidic or genomic DNAs, proportionally to the number of repeats, suggesting that longer repeats tend to form more frequently imperfect hairpins. Finally, the VHH was bound to a protein G column and used to selectively enrich structure-containing DNA molecules, that were further observed by electron microscopy. They exhibit different kinds of structures in which only one strand is folded into an imperfect hairpin. Our results unambiguously show that alternative DNA structures are transiently formed by CTG trinucleotide repeats in living bacterial and yeast cells and suggest that imperfect CTG hairpins mainly exist in vivo mainly as heteroduplex DNA molecules.

molecular biology↗

bioRxiv2023

The Bacterial Replicative Helicase Loader DciA is a DNA Condenser.

The loading of the bacterial replicative helicase is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that VcDciA from Vibrio cholerae, composed of a globular KH-like domain followed by an unfolded extension, has a strong affinity for DNA. Here, we characterized the droplets formed by VcDciA upon interaction with a short single-stranded substrate. We demonstrate the fluidity of these droplets using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of VcDnaB inside the VcDciA-DNA droplets. We show that DnaC from Escherichia coli is also competent to form these condensate structures in the presence of ssDNA. Our data open up possibilities for the involvement of DciA in the formation of non-membrane compartments within the bacterium, facilitating the assembly of replication players with the chromosomal DNA.

biochemistry↗

bioRxiv2022

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist SpRecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATP{gamma}S. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

microbiology↗

bioRxiv2022

Human RAD52 stimulates the RAD51-mediated homology search

Homologous recombination (HR) is a DNA repair mechanism of double strand breaks and blocked replication forks that involves a process of homology search and homologous pairing leading to the formation of synaptic intermediates whose architecture and dynamics are tightly regulated to ensure genome integrity. In this mechanism, RAD51 recombinase plays a central role and is supported by many partner including BRCA2 and RAD52. If the mediator function of BRCA2 to load RAD51 on RPA-covered ssDNA is well established, the role of RAD52 in HR, more precisely its interplay with BRCA2 is still far from understood. We have used Transmission Electron Microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52 and BRCA2 in the assembly of the RAD51 filament, its architecture and its activity. Despite our results confirm that RAD52 lacks a mediator activity, we observed that RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator role of BRCA2 and form shorter RAD52- and RAD51-containing mixed filaments that are more efficient in subsequent homology search and formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. Through the characterization of the behavior of RAD52 and BRCA2, these results provide new molecular insights on the formation and regulation of presynaptic and synaptic intermediates during human HR.

biochemistry↗

bioRxiv2022

In vivo inactivation of RAD51-mediated homologous recombination leads to premature aging, but not to tumorigenesis

Genetic instability is a hallmark of both cancer and aging. Homologous recombination (HR) is a prominent DNA repair pathway maintaining genomic integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the consequences of mutations in the pivotal HR player, RAD51, on cancer development remain puzzling. Moreover, in contrast with other HR genes, RAD51 mouse models are not available to experimentally address the role of RAD51 on aging and carcinogenesis, in vivo. Here, we engineered a mouse model with an inducible dominant negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative non-conservative repair pathways. We found that, in vivo expression of SMRad51 did not trigger tumorigenesis, but instead induced premature aging. We propose that these in vivo phenotypes result from the exhaustion of proliferating progenitors submitted to chronic endogenous replication stress resulting from RAD51-mediated HR suppression. Our data underline the importance of the RAD51 activity for progenitors homeostasis, preventing aging, and more generally for the balance between cancer and aging.

cancer biology↗

bioRxiv2020

Replication intermediate architecture reveals the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase{eta} , able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of pol{eta} in shaping this response and ensuring the continuity of DNA synthesis.

molecular biology↗

bioRxiv2019

Mammalian RAD51 prevents non-conservative alternative end-joining and single strand annealing through non-catalytic mechanismssms

The selection of the DNA double-strand breaks (DSBs) repair pathway is decisive for genetic stability/instability. We proposed that it acts according to two successive steps: 1-canonical non-homologous end-joining (C-NHEJ) versus single-strand DNA (ssDNA) resection; 2- on ssDNA, gene conversion (GC) versus non-conservative single-strand annealing (SSA) or alternative end-joining (A-EJ).\n\nUsing intramolecular substrates, we systematically analysed the equilibrium between the different DSB repair pathways. We show that ablation of RAD51 stimulated both SSA and A-EJ but did not stimulate C-NHEJ, validating the two-step model. Moreover, we found that two ATP-mutant dominant-negative forms of RAD51 that stimulated non-conservative repair, failed to load into damaged chromatin, clarifying the role of ATP in RAD51-mediated HR, also. In contrast, another dominant-negative form of RAD51, which retains its DNA binding capacities, repressed SSA and A-EJ, revealing two separable functions of RAD51 i.e. GC and non-conservative repair inhibition. In vitro assays show that the binding of RAD51 on both complementary ssDNA is required to block both spontaneous and RAD52-induced strand annealing. Therefore, RAD51 represses non-conservative repair (SSA and A-EJ), by inhibiting the annealing step through ssDNA occupancy, independently of the catalytic strand-exchange activity required for GC.

molecular biology↗