Since their discovery, R-loops have been associated with both physiological and pathological functions that are conserved across species. R-loops are a source of replication stress and genome instability, as seen in neurodegenerative disorders and cancer. In response, cells have evolved pathways to prevent R-loop accumulation as well as to resolve them. A growing body of evidence correlates R-loop accumulation with changes in the epigenetic landscape. However, the role of chromatin modification and remodeling in R-loops homeostasis remains unclear. This review covers various mechanisms precluding R-loop accumulation and highlights the role of chromatin modifiers and remodelers in facilitating timely R-loop resolution. We also discuss the enigmatic role of RNA:DNA hybrids in facilitating DNA repair, epigenetic landscape and the potential role of replication fork preservation pathways, active fork stability and stalled fork protection pathways, in avoiding replication-transcription conflicts. Finally, we discuss the potential role of several Chro-Mates (chromatin modifiers and remodelers) in the likely differentiation between persistent/detrimental R-loops and transient/benign R-loops that assist in various physiological processes relevant for therapeutic interventions.

Fine-tuning DNA replication and transcription is crucial to prevent collisions between their machineries¹. This is particularly important near promoters, where RNA polymerase II (RNAPII) initiates transcription and frequently arrests, forming R-loops^2,3,4. Arrested RNAPII can obstruct DNA replication, which often initiates near promoters^5,6. The mechanisms that rescue arrested RNAPII during elongation to avoid conflicts with co-directional replisomes remain unclear. Here, using genome-wide approaches and genetic screens, we identify CFAP20 as part of a protective pathway that salvages arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops near promoters, which leads to defects in replication timing and dynamics. These defects stem from accelerated replication-fork speeds that cause a secondary reduction in origin activity. Co-depletion of the Mediator complex or removal of R-loop-engaged RNAPII restores normal replication. Our findings suggest that transcription-dependent fork stalling in cis induces accelerated fork progression in trans, generating single-stranded DNA gaps. We propose that CFAP20 facilitates RNAPII elongation under high levels of Mediator-driven transcription, thereby preventing replisome collisions. This study provides a transcription-centred view of transcription–replication encounters, revealing how locally arrested transcription complexes propagate genome-wide replication phenotypes and defining CFAP20 as a key factor that safeguards genome stability.

Transcription–replication conflicts (TRCs) arise when DNA replication forks encounter actively transcribing RNA polymerases, creating a major threat to genome stability. These conflicts, which can occur in different orientations, disrupt replication fork progression, impair transcriptional fidelity and reshape the chromatin landscape. In this review, we discuss emerging conceptual insights into how cells coordinate replication and transcription in space and time to minimise such encounters, and we highlight the central role of RNA polymerase II dynamics in both preventing and resolving TRCs. We further describe how TRCs engage a broad network of genome maintenance pathways that regulate R-loops, stabilize stalled forks and maintain chromatin integrity. Importantly, elevated or mismanaged TRCs create vulnerabilities that many cancers exploit, positioning conflict-resolution mechanisms as attractive therapeutic targets. Finally, we examine current challenges in detecting and analysing these transient, dynamic events and underscore the need for improved imaging and sequencing technologies to study the genome’s molecular “traffic jams”. A deeper mechanistic understanding of TRCs will be crucial for harnessing them in precision oncology and clarifying their broader roles in genome regulation.

In this study, we sought to comprehensively map ubiquitin signaling networks at the crossroads of cellular proliferation and proteostasis, both crucial in cancer. Genome-wide CRISPR knockout screening in the presence of the ubiquitin E1 inhibitor TAK-243 identified 239 genes whose ablation sensitizes cells to ubiquitination blockade. Follow-up studies reveal a role for the nuclear protein, C16orf72/HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1)–Associated Protein modifying Stress Responses (HAPSTR1), in chromatin proteostasis and DNA replication. We show that HAPSTR1 recruits the E3 ligase HUWE1 to the chromatin, keeping the chromatin-associated proteome in check, and is itself turned over by HUWE1 during cell cycle progression. Structural modeling of the HAPSTR1/HUWE1 complex, supported by mutational analysis, demonstrates that loss of HUWE1 binding renders HAPSTR1 unable to support cellular proliferation when ubiquitin signaling is suppressed. Consequently, HAPSTR1 ablation impairs clearance of chromatin-associated proteins, triggering global transcriptional and proteostatic adaptations. Under diminished ubiquitin signaling, these adaptive mechanisms give way to replication stress and premature S phase entry, culminating in cell cycle collapse and DNA damage. Our findings uncover protein networks exhibiting synthetic lethality with ubiquitin signaling blockade and highlight vulnerabilities of actively proliferating cells to proteostatic imbalance at the chromatin.