Biology subjects

Garcia-Rodriguez, N.

Publications and source records attributed to Garcia-Rodriguez, N..

6 recordsLinked to original sources

bioRxiv2026

RNF25 Ubiquitin E3 activity Safeguards Genome Integrity by Modulating DNA Replication, Transcription and Translation.

The human genome encodes several hundreds of ubiquitin E3 enzymes, most of which have poorly understood biological roles. In search for novel ubiquitin E3 enzymes involved in DNA damage tolerance, we identified RNF25 as a candidate to have a role in DNA replication stress tolerance. Under stress conditions, RNF25 translocates to the nucleus in a cGAS-dependent manner, and loss of RNF25 ubiquitin E3 activity leads to the accumulation of replication-dependent ssDNA gaps. Combining functional assays with with mass-spectrometry based proteomics approaches such as TULIP2, iPOND-MS and TurboID, we found that, mechanistically, RNF25 promotes the stability of the replication fork at Transcription-Replication Conflicts by the ubiquitin-mediated clearance of RAD18, mono-ubiquitinated PCNA, H2B-K120ub and RECQL, among others. Lack of RNF25 ubiquitin E3 activity promotes the occurrence of Transcription-Replication Conflicts and destabilizes reversed replication forks, enabling re-priming and accumulation of ssDNA gaps behind the replication fork, ultimately compromising genome integrity. Overall, RNF25 regulates DNA replication, transcription and translation in an ubiquitin-based goldilocks.

molecular biology↗

bioRxiv2026

H3K37me1 couples transcription with DNA replication origin selection

DNA replication initiation in higher eukaryotes occurs at thousands of sites distributed throughout the genome and follows a defined temporal program. However, replication origins are not determined by a conserved DNA motif, and how specific genomic regions are selected for initiation remains poorly understood. Here, we identify a transcription-associated histone modification in human cells, mono-methylation of histone H3 lysine 37 (H3K37me1), and show that it regulates the spatial organization of replication initiation. H3K37me1 is enriched across actively transcribed gene bodies, and its depletion leads to a redistribution of replication origin activity toward intragenic regions. We show that H3K37me1 limits the association of the MCM2-7 replicative helicase with transcribed chromatin, thereby restricting intragenic origin usage. Consistently, loss of H3K37me1 increases both MCM2-7 occupancy and replication initiation across gene bodies. Collectively, our findings uncover a chromatin-based mechanism that couples transcription with DNA replication origin selection by limiting unscheduled origin firing within transcribed regions.

Molecular Biology↗

bioRxiv2025

miRNA-mediated cell-to-cell communications boost DNA repair during theRadioadaptative Response

The Radioadaptive Response (RAR) is a phenomenon where a low, or priming, dose of ionizing radiation enhances cellular resistance to subsequent higher doses. We investigated whether RAR involves alterations in Homologous Recombination (HR), a high-fidelity DNA repair pathway. Using fibroblast models, we found that primed cells exhibit accelerated DNA end resection, an initial and essential HR step. This effect is mostly mediated by a bystander mechanism involving small extracellular vesicles (sEVs), as conditioned media fully replicated it. RNA profiling of sEVs identified miR-126-3p and miR-451a as key regulators of this response. Significantly, inhibiting miR-451a induced RAR in normally unresponsive cells. We further identified a miR-451a-p38-CCAR2 axis that enhances HR through suppression of CCAR2. These findings delineate a novel miRNA-mediated, sEV-driven mechanism that regulates HR during RAR, with potential therapeutic implications. Significance StatementWe uncover a previously unrecognized mechanism by which human fibroblasts enhance DNA double-strand break repair through homologous recombination following a priming dose of ionizing radiation-a phenomenon known as the radioadaptive response (RAR). We demonstrate that this enhanced repair capacity is driven by small extracellular vesicle (sEV)-mediated intercellular communication, through a transient reprograming of the DNA repair capacity of neighboring cells by modulating the levels of two key microRNAs. These provide new clues on how extracellular RNA signaling governs genome maintenance, with significant implications for genome stability in healthy and pathological context. The identification of actionable modulators further strengthens the translational potential of our work.

molecular biology↗

bioRxiv2024

EXO1-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells.

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Furthermore, we show that EXO1-deficient cells exhibit marked sensitivity to translesion synthesis inhibition, a distinctive characteristic of mutations in proteins essential for repairing ssDNA gaps via template switching, such as BRCA1/2. Strikingly, EXO1 loss results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. Indeed, BRCA1-deficient cells become addicted to the overexpression of EXO1 DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

molecular biology↗

bioRxiv2023

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis.

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. We found that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes replication fork stability epistatically to BRCA1 S114 phosphorylation, addressing the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.

molecular biology↗

bioRxiv2021

Manganese is a Physiologically Relevant TORC1 Activator in Yeast and Mammals.

The essential biometal manganese (Mn) serves as a cofactor for several enzymes that are crucial for the prevention of human diseases. Whether intracellular Mn levels may be sensed and modulate intracellular signaling events has so far remained largely unexplored. The highly conserved target of rapamycin complex 1 (TORC1, mTORC1 in mammals) protein kinase requires divalent metal cofactors such as magnesium (Mg2+) to phosphorylate effectors as part of a homeostatic process that coordinates cell growth and metabolism with nutrient and/or growth factor availability. Here, our genetic approaches reveal that TORC1 activity is stimulated in vivo by elevated cytoplasmic Mn levels, which can be induced by loss of the Golgi-resident Mn2+ transporter Pmr1 and which depends on the natural resistance-associated macrophage protein (NRAMP) metal ion transporters Smf1 and Smf2. Accordingly, genetic interventions that increase cytoplasmic Mn2+ levels antagonize the effects of rapamycin in triggering autophagy, mitophagy, and Rtg1-Rtg3-dependent mitochondrion-to-nucleus retrograde signaling. Surprisingly, our in vitro protein kinase assays uncovered that Mn2+ activates TORC1 substantially better than Mg2+, which is primarily due to its ability to lower the Km for ATP, thereby allowing more efficient ATP coordination in the catalytic cleft of TORC1. These findings, therefore, provide both a mechanism to explain our genetic observations in yeast and a rationale for how fluctuations in trace amounts of Mn can become physiologically relevant. Supporting this notion, TORC1 is also wired to feedback control mechanisms that impinge on Smf1 and Smf2. Finally, we also show that Mn2+-mediated control of TORC1 is evolutionarily conserved in mammals, which may prove relevant for our understanding of the role of Mn in human diseases. Significance StatementThe target of rapamycin complex 1 (TORC1, mTORC1 in mammals) is a central, highly conserved controller of cell growth and aging in eukaryotes. Our study shows that the essential biometal manganese (Mn) acts as a primordial activator of TORC1 and that NRAMP metal ion transporters control TORC1 activity by regulating cytoplasmic Mn2+ levels. Moreover, TORC1 activity regulates Mn2+ levels through feedback circuits impinging on NRAMP transporters. Altogether, our results indicate that Mn homeostasis is highly regulated and modulates key cellular processes such as autophagy, mitophagy, and Rtg1-3 complex-dependent retrograde response. These findings open new perspectives for the understanding of neurodegenerative disorders and aging-related processes

cell biology↗