Biology subjects

Casajus-Pelegay, E.

Publications and source records attributed to Casajus-Pelegay, E..

4 recordsLinked to original sources

bioRxiv2026

Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Phosphatidylinositol 3-kinase-related kinases (PIKKs), including ATM, ATR, DNA-PKcs, SMG1, TRRAP and mTOR, are regulators of gene expression, DNA damage response and metabolism. Their maturation depends on R2TP, a co-chaperone complex comprising the RUVBL1/2 AAA+ ATPase, RPAP3 and PIH1D1. While small-molecule RUVBL1/2 inhibitors have shown promise in cancer models, the presence of RUVBL1/2 in other complexes raises concerns about specificity. Here, we identified the RUVBL1/2-RPAP3 interface as a vulnerability, exploiting it using de novo designed protein binders that occupy the RPAP3-binding site. These subnanomolar-affinity binders dismantle R2TP, depleting levels of all PIKKs while sparing other RUVBL1/2-containing complexes, including chromatin remodelers INO80, SRCAP and TIP60. CryoEM, proteomic, biochemical and cellular analyses revealed that these binders outcompete RPAP3, trapping RUVBL1/2 in a catalytically inactive conformation. Our findings establish selective disruption of R2TP by artificial proteins as a strategy to control PIKK abundance.

molecular biology↗

bioRxiv2026

A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks

Mitochondrial DNA (mtDNA) maintenance is essential for cellular homeostasis, and defects in mtDNA replication are linked to a broad spectrum of mitochondrial diseases. During replication, DNA polymerase {gamma} (Pol{gamma}) must traverse duplex junctions and stable secondary structures, yet how the human enzyme overcomes these barriers remains incompletely understood. Here, cryo-electron microscopy structures of Pol{gamma} bound to forked DNA, G-quadruplex (G4)-containing DNA and DNA bound to mitochondrial single-stranded DNA-binding protein (mtSSB) reveal a common template-entry path along the catcher domain across distinct substrate and active-site configurations. Within this domain, an arginine-rich helix containing R1026, R1030 and R1034 constitutes a Template Stabilising Motif (TSM). Biochemical reconstitution and DNA-binding experiments show that disrupting the TSM selectively impairs strand displacement, RNA-DNA hybrid displacement and synthesis through G4-forming sequences, while largely preserving synthesis on unstructured templates. Single-molecule optical-tweezers experiments further show that mechanical destabilisation of the fork partially restores mutant strand-displacement activity, whereas force or mtSSB restores primer-extension kinetics on ssDNA templates. Together, these findings establish the role of the TSM in maintaining productive template engagement and identify template stabilisation as a common mechanism enabling Pol{gamma} to traverse structurally diverse barriers in mtDNA.

molecular biology↗

bioRxiv2024

A comprehensive genetic catalog of human double-strand break repair

The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. By employing parallel in-pool profiling of Cas9-induced indels within a genome-wide knockout library, we present a comprehensive catalog detailing how virtually every human gene influences the DSB repair process. This REPAIRome resource is validated through the identification of novel mechanisms, pathways and factors involved in DSB repair, including unexpected opposing roles for XLF and PAXX in DNA end processing, a molecular explanation for Cas9-induced multi-nucleotide insertions, the identification of HLTF as a DSB-repair factor, the involvement of the SAGA complex in microhomology-mediated end joining, and importantly, an indel mutational signature linked to VHL loss, renal carcinoma and hypoxia. Collectively, these results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.

molecular biology↗

bioRxiv2023

An ATF4-centric regulatory network is required for the assembly and function of the OXPHOS system

Identifying the factors that determine mammalian cell viability when oxidative phosphorylation (OXPHOS) function is impaired poses challenges due to the diverse cellular responses and limited clinical material availability. Moreover, animal models often fail to replicate human phenotypes. To address these challenges, this study conducted comprehensive analyses involving multiple defects and species by comparing the RNA-Seq expression profiles of human and murine cell lines with distinct nuclear backgrounds, representing both normal and OXPHOS-deficient models. To minimize species-specific variation, the study employed clustering techniques to group murine genes affected by OXPHOS dysfunction and identified crucial regulators like ATF4, UCP1, and SYVN1. ATF4 consistently displayed activation in response to OXPHOS defects, not only in murine but also in human cells, confirming its pivotal role in the cellular response to mitochondrial dysfunction. By integrating human and murine data, the study unveiled a conserved regulatory network encompassing genes related to the mTOR pathway and folate metabolism. Remarkably, the study uncovered an unexpected finding: the depletion of ATF4 in both mouse and human cells impairs OXPHOS assembly and supercomplex organization. This impairment primarily stems from a severe disruption in complex I assembly in the absence of ATF4, even under non-stress conditions.

molecular biology↗