Biology subjects

Veaute, X.

Publications and source records attributed to Veaute, X..

10 recordsLinked to original sources

bioRxiv2026

Unveiling a missing component of the atypical type IV secretion system required for natural transformation of Helicobacter pylori

Exchange of genetic information by natural transformation shapes bacterial evolution. In Helicobacter pylori it is thought to drive its unusually high recombination rate, which has a crucial role in the evolution of virulence and the propagation of antibiotics resistance genes. While in most cases uptake of the incoming DNA into the periplasm is mediated by type IV pili, in H. pylori this initial step of natural transformation requires ComB, a unique competence-specific type IV secretion system (T4SS). The mechanisms by which ComB mediates DNA uptake are still poorly understood, since T4SS are usually involved in an opposite process of DNA export. Here, we identify a gene (hp1421) that is absolutely required for uptake of the transforming DNA into the periplasm, although distant from the comB operons. We show that hp1421 codes for a hexameric ATPase from the VirB11 family. HP1421 is present in the cytoplasm and interacts with ComB4, another ATPase of the T4SS inner membrane subcomplex. The structural modelling and functional analysis of HP1421 and its interaction with ComB4 indicate that HP1421 is a missing component of the ComB inner-membrane subcomplex that we propose to name ComB11. Phylogenetic analyses show that comB11 is a H. pylori core gene and suggest that the competence-dedicated ComB T4SS was a recent acquisition within Helicobacteraceae. Hence, co-option of the T4SS for DNA transformation requires nearly all the proteins that were previously essential for DNA conjugation. Author SummaryThe capacity of bacteria to exchange genetic information contributes in the case of pathogens to the spreading of antibiotic resistance and virulence factors. For Helicobacter pylori, a Gram-negative pathogen that colonises about half of the world population and is at the origin of diseases such as ulcers and gastric cancers, natural transformation is the major mechanism of horizontal gene transfer. However, H. pylori uses a very unusual system to capture and internalise the foreign DNA. Indeed, a Type 4 secretion system mediates this process. Here, we identify a so far missing and essential component of the T4SS, coded by a gene distant from the operon coding the other subunits. Through a combination of structural modelling, biochemical and microscopy approaches we show that this ATPase is an indispensable part of the ComB T4SS. Our study provides new insights into the mechanism by which the peculiar ComB T4SS works backwards to allow the passage of the tDNA from the bacterial environment into the periplasm.

microbiology↗

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

Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

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 the dissociation of Rad51 filaments 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, preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and protecting Rad51 filaments from destabilization by Srs2.

molecular biology↗

bioRxiv2023

Telomere protein arrays stall DNA loop extrusion by condensin

DNA loop extrusion by SMC proteins is a key process underlying chromosomal organization. It is unknown how loop extruders interact with telomeres where chromosome ends are covered with a dense array of tens of neighboring DNA-binding proteins. Using complementary in vivo and in vitro single-molecule approaches, we study the interaction between loop-extruding condensin and arrays of Rap1, the double-stranded-DNA-binding telomeric protein of Saccharomyces cerevisiae. We show that dense linear Rap1 arrays can completely halt DNA loop extrusion, where the blocking efficiency depends on the array length and the DNA gap size between neighboring proteins. In cells, Rap1 arrays in the chromosome are found to act as contact insulators and to accumulate condensin at their borders, with direct implications for the resolution of dicentric chromosomes produced by telomere fusions. Our findings show that linear arrays of DNA-bound proteins can efficiently halt DNA loop extrusion by SMC proteins, which may impact a wide range of cellular processes from telomere functions to transcription and DNA repair.

molecular biology↗

bioRxiv2023

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication

RNA-binding proteins are found at replication forks, but their direct interaction with DNA-embedded RNA species that inevitably shape physiological DNA replication remains unexplored. Here we report that 53BP1, involved in the DNA damage and replication stress response, is an RNA-binding protein that directly interacts with Okazaki fragments, in the absence of any external stress. The bulk chromatin association of 53BP1 shows dramatic dependence on PRIM1, which synthesizes the RNA primer of Okazaki fragments. The direct recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment. Conversely, depletion of FEN1, which results in the accumulation of uncleaved RNA primers, leads to an upregulation of 53BP1 levels at the replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1, distinct from its canonical mode of chromatin-binding, is anchored at the replication fork through its RNA-binding activity, highlighting the role of an RNA-protein interaction at DNA replication forks.

molecular biology↗

bioRxiv2022

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA scanning and recruitment to oxidized bases in living cells

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly scans the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This scanning process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the scanning of the DNA and recruitment to oxidative lesions.

cell biology↗

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↗

bioRxiv2021

ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram-positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

microbiology↗

bioRxiv2021

Sir3 Heterochromatin Protein Promotes NHEJ by Direct Inhibition of Sae2

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) complex assembles heterochromatin-like chromatin at subtelomeres. SIR-mediated repressive chromatin limits double strand break (DSB) resection protecting damaged chromosome ends during HR. As resection initiation marks the cross-road between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR- mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Sir3 physically interacts with Sae2 and this interaction impairs Sae2-dependent MRX functions. As a consequence, Sir3 limits Mre11-mediated resection, delays MRX removal from DSB ends and promotes NHEJ.

genetics↗

bioRxiv2018

MEIOB and SPATA22 resemble RPA subunits and interact with the RPA complex to promote meiotic recombination

Homologous recombination is a conserved DNA repair process mandatory for chromosome segregation during meiosis. RPA, a ubiquitous complex essential to recombination, is thought to play a similar role during mitotic and meiotic recombination. MEIOB, a meiosis-specific factor with unknown molecular function, ressembles a RPA subunit. Here we use in vivo approaches to show that in mouse spermatocytes, DMC1 and RAD51 appear to be normally loaded in the absence of MEIOB but are prematurely lost from unrepaired recombination sites. This loss correlates with an accumulation of the BLM helicase on meiotic chromosomes. We also show that MEIOB alters the immunodetection of RPA subunits at meiotic recombination sites. Using electron microscopy and purified proteins, we demonstrate that the MEIOB-SPATA22 complex associates with and modifies the conformation of RPA-coated ssDNA. Finally, we identify structural homology between MEIOB, SPATA22 and RPA subunits, and show that MEIOB and SPATA22 interact through C-terminal OB-fold containing domains (OBCDs) like RPA subunits. Moreover, MEIOB and SPATA22 cooperate to interact with RPA through their OBCDs. Our results suggest that MEIOB, SPATA22 and RPA work together to ensure proper processing of meiotic recombination intermediates.

cell biology↗