Molecular Cell

Regulation of protein synthesis is essential for maintaining cellular homeostasis during stress. The integrated stress response (ISR) is a conserved signaling pathway that modulates global mRNA translation through four eIF2 kinases--GCN2, PKR, PERK, and HRI. However, how these kinases are selectively activated and tuned to distinct stress signals to direct appropriate cell fate decisions remains poorly understood. Here, we employ ultra-deep mutagenesis screens to systematically map regulators of protein synthesis across diverse stress perturbations in human cells. This comparative approach identifies stress-specific translational control factors, including a previously unrecognized role for the E3 ubiquitin ligase RNF25 in selectively sustaining translation following UV irradiation and other RNA-damaging treatments. In this context, we demonstrate that RNF25 operates independently of its partner RNF14, and that its ubiquitin ligase activity, as well as its RWD-domain, is required to restrain excessive activation of the eIF2 kinase GCN2. Accordingly, loss of RNF25 results in hyperactivation of GCN2, exacerbated translation shutdown, and impaired cell proliferation following RNA damage--phenotypes that can be fully reversed by genetic or pharmacological inhibition of GCN2. Together, these findings uncover a previously unappreciated RNF25-GCN2 signaling axis and identify ISR-driven toxicity as a potential vulnerability in combination with RNA-damaging chemotherapeutics.

PDS5A, a regulatory subunit of the cohesin complex, and topoisomerase IIB (TOP2B), an enzyme resolving DNA topological problems, interact with CTCF and regulate transcription, chromatin loops, and genome organization. Yet, how PDS5A and TOP2B are recruited to chromatin to exert their function is not well-understood. Here, we studied the functional relationship between PDS5A and TOP2B and the resultant impact on genome organization and gene expression. Interestingly, TOP2B-PDS5A cooperate for their recruitment to CTCF-bound chromatin sites. The presence of catalytically active TOP2B increased PDS5A occupancy genome-wide. Notably, a novel PDS5A-CTCF interaction region in the CTCF N-terminal 95-116aa was required for CTCF-PDS5A-TOP2B interaction in vitro as well as for active TOP2B-mediated enrichment of PDS5A chromatin occupancy in vivo. The loss of CTCF(95-116aa) led to a reduced number of chromatin loops and dysregulated gene expression. In gliomas, PDS5A and TOP2B expression levels are variable and correlated, contributing to apparent heterogeneity in gene expression. Indeed, inducible knockdown of PDS5A led to reduced TOP2B occupancy and altered gene expression in the glioma genome. Importantly, PDS5A mediated sensitivity to TOP2 cancer drugs in glioma cells. This newly recognized functional interaction between PDS5A and TOP2B at chromatin boundaries clarifies the mechanisms fostering gene regulation through genome organization, with implications for glioma therapeutics.

DNA replication initiation requires activation of the CMG helicase to establish the replisome. This process involves the extrusion of single-stranded DNA (ssDNA) from the central channel of MCM double hexamers, allowing the two CMG helicases to pass each other; however, the factors that mediate this process in human cells remain unclear. We show that degron-mediated depletion of either MCM10 or RECQL4 alone causes only mild replication defects, whereas simultaneous depletion of both proteins completely blocks CMG activation. ChIP-seq analyses demonstrate that RECQL4 localises to replication initiation zones (IZs) independently of MCM10, whereas MCM10 recruitment to IZs is enhanced upon RECQL4 depletion, suggesting RECQL4 primarily functions in CMG activation, and MCM10 acts as a backup or supporting factor. Rescue experiments further indicate that RECQL4 cooperates with MCM10 through direct interaction, and that their ssDNA-binding activity underlies their functional overlap. We propose MCM10 and RECQL4 act cooperatively and redundantly to promote CMG activation.

During development, cellular identity is ultimately determined by transcriptional output: lineage-specific genes must be activated, while genes associated with alternative fates must be repressed. This process depends on the activity of chromatin remodelling complexes, which regulate the accessibility of transcription factors to chromatin regulatory elements. In addition, cellular identity is shaped by exposure to intercellular signals. Understanding the mechanisms by which extracellular signals are translated into changes in the transcriptional program is essential for understanding cell fate decisions during development, as well as in disease conditions such as cancer. Here we describe a rapid and widespread enhancer resetting event in response to ERK signalling in mouse ES cells. This process occurs in two distinct phases: an immediate, genome-wide alteration in transcription factor binding dynamics at regulatory regions which is dependent on the release of paused RNA Polymerase II, followed by the re-establishment of a context appropriate, stable chromatin state. We demonstrate that the chromatin remodelling complex NuRD is required for this reestablishment phase and for the appropriate transcriptional response to ERK signalling. We propose that enhancer resetting places genomic regulatory regions in a state which is permissive to the exchange of transcription factors in order to establish a new, stable enhancer topology enabling rapid yet precise transcriptional response to extracellular signals.

TDP-43 is a nuclear RNA-binding protein regulating numerous steps in RNA metabolism, including alternative splicing. It is a major pathological hallmark of several neurodegenerative diseases, where it forms cytoplasmic aggregates in affected brain regions. TDP-43 can undergo phase separation (PS) and this condensation behavior may be linked to aggregate formation. Whether and how PS governs TDP-43s RNA regulatory functions remains poorly understood. Here we utilized rationally designed mutations in the TDP-43 low complexity domain to tune TDP-43 PS, yielding a panel of TDP-43 variants with reduced propensity to form condensates ("PS-deficient"), and a panel of TDP-43 variants that form more irreversible, undynamic condensates ("solid-like") in vitro and in cells. Affinity proteomics coupled to mass spectrometry identified a set of interactors whose association with TDP-43 is PS-dependent. This includes multiple splicing regulators and the RNA helicase UPF1, which show increased interactions with solid-like TDP-43 variants. Consistently, we identified PS-dependent alternative splicing events that translate into measurable changes in RNA and protein abundance. Our results highlight that TDP-43 PS regulates RNA and protein homeostasis both directly, by altering a subset of TDP-43-dependent alternative splicing events, and indirectly, by changing interactions with other RNA regulatory factors.

Casein kinase 1{delta} (CK1{delta}) is a ubiquitously expressed kinase involved in diverse cellular processes, including cell cycle regulation. CK1{delta} activity is attenuated by (auto)phosphorylation. However, inhibitory phosphorylation is efficiently opposed by cellular phosphatases as CK1{delta} accumulates in its hypophosphorylated, active state. CK1{delta} is a target of the nuclear ubiquitin ligase APC/C-CDH1, yet the kinase is apparently stable. Thus, the physiological relevance of CK1{delta} (auto)phosphorylation, autoinhibition, and regulated turnover has remained unclear. Here we show that CK1{delta} activity and abundance are coordinated in a cell cycle-dependent manner. During G1, assembled CK1{delta} kinase is stable while free active kinase is degraded. In S phase, unassembled CK1{delta} is no longer degraded, likely to support functions in DNA damage signaling. Upon mitotic entry, the downregulation of phosphatases promotes CK1{delta} (auto)phosphorylation and consequent autoinhibition, thereby preserving a pool of kinase to rapidly reestablish the post-mitotic steady state.

The FET family of RNA-binding proteins, FUS, EWSR1, and TAF15, contribute to transcriptional regulation and RNA maturation, but their core functions remain unclear. Chromosomal rearrangements involving FUS, EWSR1, or TAF15 drive multiple cancers, and mutations in the genes encoding the FET proteins are associated with neurodegenerative disease. Here, using nanoscale imaging, we show that endogenous EWSR1 and newly synthesized RNA exhibit a network-like organization with EWSR1 foci forming the nodes of this ribonucleoprotein network. Acute depletion of EWSR1 causes a rapid but transient reduction in nascent RNA levels and cellular metabolic activity without affecting active transcription. Notably, loss of EWSR1 induces a compensatory mechanism involving the reorganization of FUS and TAF15 to closely resemble that of EWSR1, including enhanced clustering with newly synthesized RNA. Together, our findings reveal functional redundancy within the FET protein family that is critical for the homeostatic regulation of nascent RNA levels. In briefSundara Rajan et al. show that endogenous EWSR1 and nascent RNA form a ribonucleoprotein network. EWSR1 depletion transiently reduces nascent RNA and metabolic activity without impairment of transcriptional elongation. Loss of EWSR1 induces compensatory reorganization of FUS and TAF15, revealing a protein family mechanism required for the homeostatic regulation of nascent RNA levels. HighlightsO_LIEWSR1 and nascent RNA form a ribonucleoprotein network C_LIO_LIEWSR1 loss transiently reduces nascent RNA and metabolic activity C_LIO_LIFUS and TAF15 undergo compensatory nuclear reorganization upon EWSR1 loss C_LIO_LIFUS and TAF15 functionally replace EWSR1 C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/713985v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@b66671org.highwire.dtl.DTLVardef@ff8d49org.highwire.dtl.DTLVardef@194cfd4org.highwire.dtl.DTLVardef@d889f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Quiescence is a conserved program of endurance and readiness in non-cycling cells that is fundamental to the longevity of eukaryotic lineages, from clonal microbial populations to human regenerative tissues. While this G0 state is universally associated with a compact chromatin organization, the active safeguards that preserve this structural template--and their necessity for future division competence and fidelity--remain unknown. Here we show, using fission yeast as a model, that chromatin structural maintenance in quiescence requires the enforcement of transcription termination by the conserved factor Ppn1PNUTS/Ref2. We identify a minimal disordered region in Ppn1 that resolves a physical conflict in the quiescent genome by preventing the transcriptional "eviction" of cohesin. Loss of this safeguard drives a progressive structural erosion that deregulates cyclin-dependent kinase (CDK) dynamics and causes lethal aneuploidy upon cell-cycle re-entry. Notably, this deterioration is not an inevitable terminal state; re-establishing termination via a brief Ppn1 pulse resets division fidelity by stabilizing a core de novo cohesin landscape. These findings redefine quiescent chromatin as an actively maintainable blueprint rather than a passive standby state established at G0 entry. We propose that "quiescence exhaustion" is driven by transcriptional stress eroding genomic organization, defining a structural limit to cellular longevity.

Mitosis poses major challenges to cellular transcription. As cells enter mitosis, transcription is globally silenced and must be precisely restored upon mitotic exit. These processes are primarily regulated by Cdk1-dependent phosphorylation. In parallel, additional mechanisms, including Transcription Termination Factor 2 (TTF2)-mediated removal of nascent transcripts, reinforce transcriptional shutdown. How these layers of regulation control individual RNA polymerases and influence transcriptional reactivation at mitotic exit remains poorly understood. Here, we probed how TTF2 differentially controls transcription of distinct RNA classes, using polymerase-specific perturbations and nascent RNA labelling across mitosis. Loss of TTF2 led to accumulation of chromatin-associated transcripts during metaphase, predominantly RNA Polymerase II-derived, consistent with its established role in transcriptional clearance. More unexpectedly, TTF2 depletion caused premature RNA Polymerase I reactivation during anaphase, resulting in unscheduled rRNA synthesis and early recruitment of nucleolar proteins. These findings place TTF2 as a novel regulator of RNA Polymerase I reactivation at mitotic exit. Disruption of this control persists beyond mitosis, resulting in increased nucleolar fragmentation in interphase. Together, these findings reveal TTF2 as a conserved regulator that interfaces with multiple RNA polymerases through functionally distinct modes of control, coordinating both transcriptional shutdown and timely reactivation across mitosis.

Eukaryotic genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Within this chromatin context, genomic DNA is constantly exposed to endogenous and exogenous stress that result in the formation of DNA damage, which must be effectively repaired to maintain genome stability. Single-strand breaks (SSBs) are among the most prevalent forms of DNA damage that arise via the oxidation-induced disintegration of the sugar-phosphate backbone or as repair intermediates during base excision repair. DNA ligase III (LigIII) is one of the primary enzymes responsible for repairing SSBs containing an intact 5'-phosphate and 3'-OH (nick) during the terminal step of single-strand break repair (SSBR) and base excision repair (BER) pathways. To date, a complete mechanistic description for how LigIII processes nicks within chromatin remains elusive. Here, we use a combination of biochemical assays, molecular dynamics simulations, and cryogenic electron microscopy (cryo-EM) to define the molecular basis of nick ligation in the nucleosome by LigIII. Quantitative enzyme kinetics reveal that the LigIII ligation rate is highly dependent on the translational position of the nick in the nucleosome, where nicks near the nucleosome entry/exit site are ligated with moderate efficiency and nicks near the nucleosome dyad are refractory to ligation. Cryo-EM structures of LigIII bound to nicks at four unique translational positions in the nucleosome reveal the structural basis for this position-dependent catalytic activity, identifying that local steric constraints imposed by the histone octamer prevent LigIII from readily adopting a ligation-competent conformation. Further biochemical and structural analysis demonstrates that the scaffolding protein XRCC1, which forms a heterodimer with LigIII, does not substantially alter the ability of LigIII to bind or ligate nicks in the nucleosome. Together, this work provides foundational insight into the processing of nicks in the nucleosome during the terminal step of SSBR/BER.

How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes. ImportanceThis work seeks to understand the basis for oligomerization of a large oxidoreductase that is important for metabolizing sulfur, an essential chemical for all of biology. A 52-residue long leader peptide is necessary and sufficient for assembly into a particularly stable octamer that is resistant to chemical denaturation under diverse conditions.

DNA breaks activate PARP1/2 to synthesize poly(ADP-ribose) (PAR), which relaxes chromatin and recruits DNA repair factors. Normally, PAR is short-lived, rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG). While PARP1/2 inhibitors are established therapies for homologous recombination (HR)-deficient cancers, predictive biomarkers for PARG inhibition (PARGi) remain undefined. Using parallel genome-wide CRISPR screens with PARP and PARG inhibitors, we show that PARGi is synthetically lethal with loss of several PAR-binding factors, including XRCC1-LIG3, POLB, ALC1/CHD1L, ARH3, and PARG itself, but notably not with HR deficiency. Conversely, loss of PARP1, NMNAT1 (required for nuclear NAD synthesis), or UNG (upstream of APE1 cleavage and PARP1 activation), confers PARGi resistance. Mechanistically, PARGi induces time- and dose-dependent formation of PARP1-and PAR-dependent nuclear condensates containing XRCC1 and associated repair factors in otherwise undamaged cells. These condensates do not harbor active DNA breaks but instead sequester PAR-binding repair proteins, depleting their available nuclear pool and impairing their recruitment to genuine DNA breaks. While our analysis focused on XRCC1, PARG inhibition likely sequesters additional PAR- and PARP1-binding proteins. Thus, we propose that PARGi sequesters PAR-binding proteins to elicit toxicity, explaining the essentiality of PARG (but not PARP1) and identifying the loss of PAR-binding factors as candidate predictive biomarkers for PARG-targeted therapy.

Formation of a "closed-loop" mRNP, in which the 5' cap and 3' poly(A) tail are bridged by eIF4E-eIF4G-PABP interactions, has long been proposed to drive efficient translation initiation. Direct tests of this model in mammalian cells have remained elusive. Using auxin-inducible degron (AID) technology to acutely deplete eIF4G1, we find that global translation is only partially reduced and recovers without restoration of eIF4G1 levels. We identify eIF4G3 as an underappreciated contributor to basal translation that buffers translational output upon eIF4G1 loss without increased protein expression, explaining the modest defects observed in prior RNAi-based studies. Systematic replacement of eIF4G1 with defined cleavage products and interaction mutants reveals that PABP binding by eIF4G1 is dispensable for bulk translation initiation: the central caspase-3 cleavage fragment of eIF4G1 (casp3-cpM), which lacks the PABP-interaction domain, fully rescues global protein synthesis, and acute depletion of both major cytoplasmic PABP paralogs primarily destabilizes mRNAs rather than impairing initiation. In contrast, the N-terminal enteroviral 2A cleavage product (2A-cpN) is a potent, dominant translational repressor that requires simultaneous eIF4E and PABP engagement to form a dead-end closed-loop mRNP that sequesters initiation factors without enabling 43S recruitment. These findings reveal that the eIF4G-PABP closed-loop architecture is not required for productive initiation but can be actively co-opted for translational silencing. This explains why viral eIF4G cleavage, but not factor depletion, produces near-complete translational shutoff. The modular architecture of eIF4G enables diametrically opposing translational outcomes through selective proteolytic processing.

An imbalance of DNA damage over DNA repair contributes to the genomic instability that drives aging and numerous age-related diseases. While numerous DNA repair mechanisms have been elucidated over decades of study, little is known about the contribution of metabolism to genomic stability. We report that adipose triglyceride lipase (ATGL), a primary lipolytic enzyme, promotes DNA repair. We show that lipid droplets (LDs) accumulate in response to DNA damage and that inhibition of LD biogenesis before genotoxic stress increases the persistence of DNA damage. Overexpression of ATGL (increasing lipolysis) enhances DNA repair in response to etoposide and ionizing radiation, thus reducing DNA damage burden. Mechanistically, ATGL promotes bulk acetylation of chromatin-bound proteins and blockade of the histone acetyltransferase p300 negates these effects. Further, ATGL-induced DNA repair attenuates the long-term consequences of DNA damage, and reducing senescence and enhancing viability. Overall, these studies reveal a novel role for LDs and LD proteins in DNA damage and repair, thus unveiling a mechanism through which lipid metabolism contributes to genomic stability.

Small RNA pathways provide a robust and dynamic regulatory network that enables spatiotemporal regulation of the germline genome in response to environmental cues. The flexibility of RNA interference (RNAi)-mediated gene regulation and network architecture of these pathways requires molecular mechanisms that can fine-tune their regulatory potential and function to ensure proper execution of physiological processes, such as fertility. In C. elegans, we previously discovered a set of small RNA sensors that modulate the production of one class of small RNAs to adjust amplification resources based on cellular needs. These sensors maintain homeostatic levels of 22G-RNAs for the distinct RNAi branches that compete for resources in the mutator complex. Here we show this molecular feedback is essential for restricting expression of spermatogenic transcripts to an appropriate threshold during development and preventing spermiogenesis defects. Furthermore, we demonstrate 22G-RNA homeostasis is critical for proper meiotic progression in the germline and piRNA pathway function within pachytene germ cells. Together, our work reveals that RNAi homeostasis is critical for developmental and physiological processes, such as sperm-based fertility. Further, our findings show that small RNA pathway function is more than the sum of its parts and disrupting the ability to maintain homeostasis within the regulatory pathway itself leads to deleterious physiological consequences.

The nuclear lamina plays a central role in genome organization, yet how specific lamina-associated proteins regulate chromosome architecture during development remains unclear. Here, we show that the nucleoplasmic domains of the Lamin B Receptor (LBR) are essential for X-chromosome localization at the nuclear periphery and chromatin architecture during neural differentiation. Using genetic dissection of LBR function, combined with genome-wide chromatin solubility profiling and transcriptional analyses, we demonstrate that loss of LBR N-terminal domains impairs proper cell differentiation and X chromosome inactivation (XCI), selectively disrupting chromatin structure in neural progenitors but not in pluripotent cells. Strikingly, these effects are disproportionately concentrated - but not limited to - on the inactive X chromosome, which undergoes a pronounced shift toward a more soluble chromatin state. Our findings establish the nucleoplasmic function of LBR as a key determinant of X-chromosome functionality and identify chromatin solubility and accessibility as a previously underappreciated layer of genome regulation by the nuclear lamina in XCI. Finally, our work provides definitive genetic evidence that LBRs nuclear architectural functions are molecularly separable from its metabolic sterol reductase activity, which is preserved in our model, and are critically necessary for XCI in differentiating mouse female XX ESCs models.

Translational repression enables rapid adaptation to environmental changes. Under stress, translational repressed mRNA and mRNA decay factors accumulate in cytoplasmic processing bodies (PBs), implicated in mRNA storage and decay. PBs have been mostly studied under glucose starvation in yeast, yet, knowledge is limited under other stress conditions. Here, we identify a correlation between the level of translation attenuation and the number, brightness, fluidity and recruitment of PB core components. Stresses triggering strong translation attenuation caused the formation of few bright and more fluid PBs that recruit the decay factors en bloc. Conversely, weaker translation attenuation induced numerous, dim, more viscous PBs to which PB proteins were sequentially recruited. Importantly, increasing non-translated mRNA levels augmented the brightness of dim PBs and accelerated decay machinery recruitment. Finally, boosting RNA levels increased the size of Dhh1 helicase-containing droplets in vitro. Taken together, we propose a model in which the assembly pathway and biophysical properties of PBs are governed by non-translated mRNA abundance. TeaserBiophysical properties, protein composition and assembly pathways of processing bodies are dependent on available mRNA levels.

Intron retention (IR) is a form of alternative splicing in which introns that are normally removed are retained in mature transcripts. Despite emerging evidence of widespread IR in protein-coding genes and lncRNAs, the mechanisms and functional consequences underlying this process remain poorly understood. Here, we performed a genome-wide screen, to dissect the mechanisms governing IR in the lncRNA PURPL. Unexpectedly, the top hit from the screen was the essential splicing activator U2AF2, which promotes IR in PURPL through direct binding to a weak polypyrimidine tract. Retention of this intron drives nuclear localization of PURPL and enhances cell proliferation, revealing a functional role for IR. Transcriptome-wide analysis showed that while U2AF2 promotes splicing of most transcripts, consistent with its canonical role, it also promotes IR in a distinct subset of RNAs. This subset includes the nuclear speckle localized lncRNA MALAT1, whose speckle localization is impaired upon U2AF2 depletion. Using MALAT1 knockout cells reconstituted with wild-type or intron-deleted MALAT1 variants, we identified a single intron that is essential for MALAT1 nuclear speckle localization. Deletion of this intron from endogenous MALAT1 disrupted speckle localization and reduced cell migration, phenocopying the loss of MALAT1. Together, these findings uncover a previously unrecognized role for U2AF2 in promoting intron retention and establish IR as a key mechanism regulating lncRNA localization and function.

Proper polyadenylation site (PAS) selection is critical for RNA isoform determination. Core spliceosomal components, including U1 snRNP, regulate PAS choice, but whether they work with other splicing factors in this role remains unclear. Here, we establish that the splicing factor SRSF1 regulates PAS selection independently of and through interactions with U1 snRNP. Independent of U1 snRNP, SRSF1 binds RNA near proximal PASs within 3 UTRs to promote their usage, and, in line with this observation, breast cancer tumors with altered SRSF1 levels display shifted 3'-end selection. In conjunction with U1 snRNP, SRSF1 acts on PASs through U1 snRNP-mediated SRSF1-Pol II interactions. Consistent with co-transcriptional regulation, SRSF1 reduces the Pol II elongation index and limits transcription readthrough. Together, our results reveal that SRSF1 shapes RNA isoform determination beyond its canonical role in splicing, through a combination of direct RNA binding and U1 snRNP-dependent coordination with Pol II.

Upon DNA damage, chromatin remodeling is rapidly initiated to promote chromatin accessibility, thereby facilitating the recruitment and assembly of repair factors. Although this enhanced accessibility has been linked to poly(ADP-ribose) polymerase (PARP) activity, the mechanism by which cells overcome the nucleosome barrier remains unclear. Using our designer chromatin system, we uncovered a previously uncharacterized activity of PARP1, whereby it directly and asymmetrically evicts histone dimers proximal to DNA strand breaks from nucleosomes to generate oriented hexasomes. In the presence of HPF1, PARP1 generates stable PARylated hexasomes, an open chromatin intermediate that can serve as a bifunctional hub for recruitment of DNA- and PAR-dependent factors. Using cellular assays, we demonstrated that PARP activity is both required and sufficient to drive chromatin accessibility and the recruitment of repair factors, with direct involvement of subnucleosomal species. Unexpectedly, we identified the C-terminal tail of histone H2A, a motif harboring recurrent cancer-associated mutations, as a critical determinant of efficient PARP1-mediated nucleosome disassembly. Deletion of the H2A tail sensitizes cells to DNA-damaging agents and PARP inhibitors, implicating a functional role of PARP1-mediated nucleosome disassembly in DNA repair. Together, our findings support a model in which PARP1 directly drives histone eviction, leading to the formation of subnucleosomes that facilitate efficient DNA repair.