Biology subjects

Pasero, P.

Publications and source records attributed to Pasero, P..

13 recordsLinked to original sources

bioRxiv2026

The replication checkpoint prevents fork stalling by reducing transcription

The replication checkpoint is essential for maintaining genome stability during replication stress. The prevailing view is that, without this pathway, stalled DNA replication forks lose structural integrity and collapse, preventing replication resumption and ultimately causing cell death. Here, we show that transcription is a major cause of replication stress in budding yeast checkpoint mutants. Checkpoint signaling limits replication defects by reducing RNA polymerase II and III occupancy at highly transcribed genes, particularly those encoding ribosomal proteins. Reducing transcription at these loci reduces fork stalling and the sensitivity of checkpoint mutants to hydroxyurea-induced replication stress. In human cells, transcription inhibition likewise improves replication recovery when ATR is inhibited, suggesting that this function is conserved. Together, our findings extend the canonical view of critical checkpoint functions: in addition to stabilizing stalled forks, it actively reduces RNA polymerase roadblocks that would otherwise impede their progression.

genetics↗

bioRxiv2026

The replication checkpoint couples fork progression to the release of transcribed genes from nuclear pores

The nuclear envelope contributes to chromatin compartmentalization in eukaryotes by anchoring heterochromatin to the lamina and active transcription sites to nuclear pore complexes (NPCs). During DNA replication, chromatin association with the nuclear envelope is thought to act as a topological barrier to DNA polymerases, potentially exacerbating transcription-replication conflicts (TRCs). While limited evidence suggests that the DNA replication checkpoint kinase Rad53/CHK1 promotes the release of genes from the NPC during replicative stress, the global scale and precise regulation of this process remain unknown. Through a candidate-based screen, we demonstrate that gene release from NPCs is a general feature of the cellular response to replication stress, tightly coordinated with replication fork progression. We show that the checkpoint kinases Mec1/ATR and Rad53/CHK1 are both essential for managing gene association with NPCs, but operate in a distinct, stepwise manner: Mec1/ATR triggers the initial dissociation by promoting the removal of RNAPII from the DNA, while Rad53/CHK1 prevents the premature reassociation of transcribed loci by phosphorylating the nucleoporin Mlp1. Finally, we demonstrate that maintaining this control over genes-NPC association is critical for efficient DNA replication under stress. Collectively, our findings reveal that the DNA replication checkpoint integrates genome duplication with chromosome spatial positioning to maintain genome integrity during replication stress.

genetics↗

bioRxiv2026

The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids

Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids (thereafter referred to as hybrids) could disrupt the stability and restart of stalled replication forks. The prolonged over-expression of RNase H1 is the most popular method to manipulate hybrids in situ but it cannot discriminate between direct and indirect effects resulting from their loss. To overcome this limitation, we developed iCoRD (in Cellulo RNase H Delivery) to rapidly deliver comparable amounts of either Escherichia coli RNase H1 (RnhA) or control proteins in live cells, with a negligible impact on the transcriptome. iCoRD-delivered RnhA recognises hybrids in situ, is enriched in the vicinity of active replication forks and rescues replication fork progression in different stress conditions. While iCoRD-delivered RnhA had a clear impact on stress-induced, fork-proximal hybrids and R-ChIP signals, it had no effect on DRIP or S9.6 Cut&Tag signals, even when these were induced by stress. Our results strongly suggest the existence of distinct populations of hybrids that are best mapped by different methods and are differently sensitive to RNase H1 activity in situ.

molecular biology↗

bioRxiv2025

Condensin and topoisomerases cooperate to relieve topological stress at stalled replication forks

Resolving complex topological structures at replication forks is vital for successful DNA replication, but the mechanisms are little understood. Evidence from diverse eukaryotes suggests that condensin - which promotes chromosome condensation in M phase - might also act during S phase to facilitate relaxation of torsional stress by topoisomerases. Here, we show in yeast and human cells that condensin binds stressed replication forks, where it cooperates with topoisomerases I and II to promote resection of the nascent DNA and restart replication. Our findings suggest that condensin acts with topoisomerase I at reversed forks to convert positively supercoiled DNA into structures that are subsequently relaxed by topoisomerase 2, allowing the fork to resume replication. These findings uncover an important, evolutionarily conserved role for condensin in handling topological constraints at arrested forks that is reminiscent of its function in chromosome segregation and might prevent formation of toxic chromosome structures during fork arrest and reversal.

genetics↗

bioRxiv2025

Slx4 and Fun30/SMARCAD1 coordinate S-phase checkpoint regulation and replication fork protection in response to Top1-DNA crosslinks

Replication stress is a major driver of genomic instability and is implicated in the development of diseases such as cancer. It triggers the S-phase checkpoint, a signaling pathway that coordinates the handling of replication obstacles with cell cycle progression. One prominent source of replication stress is the formation of DNA-protein crosslinks on the template, such as those induced by DNA topoisomerase I poisoning by camptothecin (CPT). In this study, we investigated how the S-phase checkpoint responds to CPT-induced replication stress. We show that both activation and timely deactivation of checkpoint signaling are critical for DNA replication completion and cell viability. Using a locus-specific approach, we found that checkpoint signaling is actively dampened at lesion sites. Mechanistically, this attenuation involves the displacement of the checkpoint mediator Rad9 by the DNA repair factors Slx4 and Fun30. This local dampening not only promotes cell cycle progression, but also permits Exo1-dependent resection of replication forks stalled by Top1-DNA crosslinks. Controlled resection, in turn, allows homologous recombination factors to access and stabilize the forks, preventing their degradation. In conclusion, we propose that local checkpoint dampening by Slx4 and Fun30 at replication stress sites is a critical mechanism that promotes replication completion and preserves genome stability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/667195v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1bdb2eorg.highwire.dtl.DTLVardef@d2d840org.highwire.dtl.DTLVardef@7366b0org.highwire.dtl.DTLVardef@5ac295_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Rapid delivery of RNase H1 enables replication fork progression under stress independently of R-loop resolution

The over-expression of RNase H1 enables the progression of replication forks under stress and safeguards genome stability. The most common interpretation of these observations is that RNase H1 over-expression removes R-loops, whose formation would hinder replication fork progression. Here we challenge this model using an innovative strategy to rapidly deliver ready-made Escherichia coli RNase H1 (RnhA) in live cells. We show that RnhA is enriched in the vicinity of active replication forks and that it rescues replication fork progression in different stress conditions. However, RnhA had no impact on R-loops mapped by the DRIP or Cut&Tag methods. Our results reveal the existence of distinct populations of RNA:DNA hybrids that are differently sensitive to RnhA and invites new interpretations of well-established observations. HIGHLIGHTSO_LIRapid delivery of R-loop sensors and regulators in live cells C_LIO_LIRapid delivery of RnhA rescues replication fork progression in stress conditions C_LIO_LIRapid delivery of RnhA does not impact R-loops C_LIO_LIDRIP detects RNA:DNA species insensitive to RNase H1 in cells C_LI

molecular biology↗

bioRxiv2023

Regulation of oncogene-induced senescence by the MRE11 and TREX1 nucleases

Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism that arrests cell proliferation in response to oncogene-induced replication stress (RS). OIS also depends on the cGAS-STING pathway, which detects cytosolic DNA and promotes the expression of type I interferons (IFN) and pro-inflammatory cytokines. Whether and how the RS and IFN responses cooperate to promote OIS is currently unknown. Here, we show that the MRE11 nuclease promotes OIS in immortalized human fibroblasts overexpressing the H-RASV12 oncogene both by slowing replication forks and by activating the cGAS-STING pathway in response to RS. Interestingly, overexpression of TREX1, the major nuclease degrading cytosolic DNA, prevented RAS-induced senescence. In contrast, overexpression of a dominant negative mutant of TREX1 was sufficient to induce senescence in human fibroblasts, even in the absence of H-RASV12 induction. Collectively, these data suggest that the RS and IFN responses in OIS are functionally linked through a process involving the nucleases MRE11 and TREX1.

molecular biology↗

bioRxiv2022

A Sterol-PI(4)P exchanger controls the Tel1/ATM axis of the DNA Damage Response

Upon DNA damage, cells activate the DNA Damage Response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. We show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidyl-4-inositol (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Further, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.

cell biology↗

bioRxiv2022

Sen1 is a master regulator of transcription-driven conflicts

Cellular homeostasis requires the coordination of several machineries concurrently engaged on the DNA. Wide-spread transcription can interfere with other processes and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase terminates non-coding transcription, but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that Sen1 removes RNA polymerase II at TRCs within genes and the rDNA, but also at sites of transcription-transcription conflicts under physiological conditions, thus qualifying as a "key regulator of conflicts". We demonstrate that genomic stability is only affected by Sen1 mutation when, in addition to its role at the replisome, termination of non-coding transcription or R-loop removal are additionally compromised.

genomics↗

bioRxiv2019

A new role of the Mus81 nuclease for replication completion after fork restart

Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway is involved in the restart of blocked replication forks. Here, we used a new method to study at the molecular level the restart of replication in response to DNA topoisomerase I poisoning by camptothecin (CPT). We show that HR-mediated restart at the global genomic level occurs by a BIR-like mechanism that requires Rad52, Rad51 and Pol32. The Mus81 endonuclease, previously proposed to cleave blocked forks, is not required for replication restart in S phase but appears to be essential to resolve fork-associated recombination intermediates in G2/M as a step necessary to complete replication. We confirmed our results using an independent system that allowed us to conclude that this mechanism is independent of the accumulation of DNA supercoiling and DNA-protein crosslinks normally caused by CPT. Thus, we describe here a specific function for Mus81 in the processing of HR-restarted forks required to complete DNA replication.

molecular biology↗

bioRxiv2017

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

CDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here we show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1, and we show that EXO1 is also required for nascent strand degradation following exposure to HU, raising the possibility that DDK regulates EXO1 directly. Thus, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.

cell biology↗

bioRxiv2017

Integrated analysis of DSB patterns reveals precisely DSB formation mechanisms following replication fork collapse

DNA double-strand breaks (DSBs) can be detected by label-based sequencing or pulsed-field gel electrophoresis (PFGE). Sequencing yields population-average DSB frequencies genome-wide, while PFGE reveals percentages of broken chromosomes. We constructed a mathematical framework to combine advantages of both: high-resolution DSB locations and their population distribution. We also use sequencing read patterns to identify replication-induced DSBs and active replication origins. We describe changes in spatiotemporal replication program upon hydroxyurea-induced replication stress. We found that one-ended DSBs, resulting from collapsed replication forks, are population-representative, while majority of two-ended DSBs (79-100%) are not. To study replication fork collapse, we used strains lacking the checkpoint protein Mec1 and the endonuclease Mus81 and quantified that 19% and 13% of hydroxyurea-induced one-ended DSBs are Mec1-and Mus81-dependent, respectively. We also clarified that Mus81-induced one-ended DSBs are Mec1-dependent.

genomics↗

bioRxiv2017

Quantitative DSB sequencing (qDSB-Seq): a method for genome-wide accurate estimation of absolute DNA double-strand break frequencies per cell

Sequencing-based methods for mapping DNA double-strand breaks (DSBs) allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. We propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induced spike-in DSBs by a site-specific endonuclease and used them to quantify labeled DSBs (e.g. using i-BLESS). Utilizing qDSB-Seq, we determined numbers of DSBs induced by a radiomimetic drug and various forms of replication stress, and revealed several orders of magnitude differences in DSB frequencies. We also measured for the first time Top1-dependent absolute DSB frequencies at replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

genomics↗