Biology subjects

Marechal, A.

Publications and source records attributed to Marechal, A..

6 recordsLinked to original sources

bioRxiv2026

Temporal proximity proteomics reveals ANP32A roles in replication fork progression and end joining

Acidic nuclear phosphoprotein 32 family member A (ANP32A) regulates chromatin and histone homeostasis, but its contribution to DNA replication and repair remains unclear. Time-resolved AirID proximity labeling with data-independent acquisition mass spectrometry revealed phased remodeling of the ANP32A proximal proteome during hydroxyurea-induced replication stress. Early stress enriched DNA replication and repair factors, including FEN1, XRCC5/Ku80, and XRCC6/Ku70, whereas prolonged exposure shifted the network towards checkpoint and cell-cycle regulators. Orthogonal proximity ligation assays revealed sustained ANP32A-FEN1 proximity and transiently increased ANP32A-XRCC5 proximity. ANP32A was dynamically recruited to {gamma}H2AX-marked lesions following laser microirradiation and showed increased proximity to {gamma}H2AX after hydroxyurea treatment. ANP32A loss impaired proliferation and G1/S progression and moderately reduced replication fork velocity. However, it did not promote nascent strand degradation at stalled forks or measurably alter homologous recombination reporter activity. In contrast, ANP32A loss reduced non-homologous end-joining reporter activity, consistent with its stress-induced proximity to Ku proteins. These findings identify ANP32A as a chromatin-associated factor supporting replication fork progression and end joining while dispensable for stalled-fork protection, linking ANP32A-dependent chromatin regulation to genome maintenance.

cell biology↗

bioRxiv2024

The Single-Stranded DNA-Binding Factor SUB1/PC4 Alleviates Replication Stress at Telomeres and is a Vulnerability of ALT Cancer Cells

AO_SCPLOWBSTRACTC_SCPLOWTo achieve replicative immortality, cancer cells must activate telomere maintenance mechanisms. In 10-15% of cancers, this is enabled by recombination-based alternative lengthening of telomeres pathways (ALT). ALT cells display several hallmarks including heterogeneous telomere length, extrachromosomal telomeric repeats and ALT-associated PML bodies. ALT cells also have high telomeric replication stress (RS) enhanced by fork-stalling structures (R-loops, G4s) and altered chromatin states. In ALT cells, telomeric RS promotes telomere elongation but above a certain threshold becomes detrimental to cell survival. Manipulating RS at telomeres has thus been proposed as a therapeutic strategy against ALT cancers. Through analysis of genome-wide CRISPR fitness screens, we identified ALT-specific vulnerabilities and describe here our characterization of the roles of SUB1, a ssDNA-binding protein, as a novel regulator of telomere stability. SUB1 depletion further increases RS at ALT telomeres, profoundly impairing ALT cell growth without impacting telomerase-positive cancer cells. During RS, SUB1 is recruited to stalled forks and ALT telomeres via its ssDNA-binding domain. This recruitment is potentiated by RPA depletion, suggesting that these factors may compete for ssDNA. The viability of ALT cells and their resilience towards RS also requires ssDNA-binding by SUB1. SUB1 depletion accelerates cell death induced by FANCM depletion, triggering unsustainable levels of telomeric damage specifically in ALT cells. Finally, combining SUB1 depletion with RS-inducing drugs rapidly induces replication catastrophe in ALT cells. Altogether, our work identifies SUB1 as a new ALT susceptibility with important roles in the mitigation of RS at ALT telomeres and suggests new therapeutic strategies for a host of still poorly managed cancers. SO_SCPLOWIGNIFICANCEC_SCPLOW SO_SCPLOWTATEMENTC_SCPLOWCurrently, there are few treatment options for ALT cancers with chemotherapy still occupying center stage despite often limited efficacy. ALT cancer cells experience high levels of replication stress at telomeres and its enhancement (e.g. via ATR inhibition) is a promising therapeutic strategy. Sensitivity to ATR inhibition varies amongst ALT cell lines/tumors warranting the development of additional ways to modulate telomeric replication stress. Here we identify SUB1, a single-stranded DNA-binding protein, as a vulnerability of ALT cells. SUB1 localizes to ALT telomeres and mitigates deleterious replication stress. SUB1 depletion synergizes with ATR inhibition and FANCM downregulation suggesting that co-targeting SUB1 with other regulators of replication stress at telomeres may kill ALT cancer cells more effectively.

cancer biology↗

bioRxiv2024

Structure of the II2-III2-IV2 mitochondrial supercomplex from the para-site Perkinsus marinus

Respiratory complexes have co-evolved into supercomplexes in different clades to sustain energy production at the basis of eukaryotic life. In this study, using cryogenic electron microscopy, we determined the 2.1 [A] resolution structure of a 104-subunit II2-III2-IV2 supercomplex from the parasite Perkinsus marinus, related to Apicomplexa, capable of complete electron transport from succinate to molecular oxygen. A feature of the parasite is the association of two copies of complex II via the apicomplexan subunit SDHG that interacts with both complexes III and IV and bridge the supercomplex. In the c1 state, we identified two protein factors, ISPR1 and ISPR2 bound on the surface of complex III, where Cytochrome c docks, acting as negative regulators. The acquisition of 15 specific subunits to complex IV results in its lateral offset, increasing the distance between the Cytochrome c electron donor and acceptor sites. The domain homologous to canonical mitochondria-encoded transmembrane subunit COX2 is made of three separate polypeptides encoded in the nucleus, and their correct assembly is a prerequisite for electron transport in the supercomplex. Subunits Cytochrome b and COX1 comprise a +2 frameshift introduced during protein synthesis by the mitoribosome. Among 114 modelled endogenous lipids, we detect a direct contribution to the formation of the divergent supercomplex and its functional sites, including assembly of CII and ubiquinone binding. Together, our findings expose the uniqueness of the principal components of bioenergetics in the mitochondria of parasites.

biochemistry↗

bioRxiv2022

Structural basis of mitochondrial membrane bending by I-II-III2-IV2 supercomplex

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that in ciliates, the membrane curvature is provided by a supercomplex containing all four respiratory chain components. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-megadalton assembly. Due to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and cristae tubulation. Our findings explain how the architecture of the native I-II-III2-IV2 supercomplex reflects the functional specialization of bioenergetics by shaping the membrane.

biochemistry↗

bioRxiv2021

POGZ modulates the DNA damage response in a HP1-dependent manner

The heterochromatin protein HP1 plays a central role in the maintenance of genome stability, in particular by promoting homologous recombination (HR)-mediated DNA repair. However, little is still known about how HP1 is controlled during this process. Here, we describe a novel function of the POGO transposable element derived with ZNF domain protein (POGZ) in the regulation of HP1 during the DNA damage response in vitro. POGZ depletion delays the resolution of DNA double-strand breaks (DSBs) and correlates with an increased sensitivity to different DNA damaging agents, including the clinically-relevant Cisplatin and Talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair pathways by retaining the BRCA1/BARD1 complex at DSBs, in a HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal and Pogz haplo-insufficiency (Pogz+/{Delta}) results in a developmental delay, impaired intellectual abilities, a hyperactive behaviour as well as a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Importantly, Pogz+/{Delta} mice are radiosensitive and accumulate DSBs in diverse tissues, including the spleen and the brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo, with clinical implications for the WHSUS.

molecular biology↗

bioRxiv2018

FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of a previously undescribed factor, FAM35A/SHDL2, as a novel effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B-cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1- and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and another previously uncharacterized protein, C20orf196/SHDL1, which promotes NHEJ and limits HR. Together, these results establish FAM35A as a novel effector of REV7 in controlling the decision-making process during DSB repair.

cancer biology↗