Significance: R-loop formation has been related to genome instability and human disease, yet the role of R-loops in replication priming remains to be explored in the eukaryotic genome. This investigation discloses that transcription-dependent R-loops have the potential to initiate origin-independent replication events in ribosomal DNA. Taken together, our data suggest that R-loops contribute to transcription-driven endoreplication events and alterations in genome copy number.

Complete and faithful duplication of its entire genetic material is one of the essential prerequisites for a proliferating cell to maintain genome stability. Yet, during replication DNA is particularly vulnerable to insults. On the one hand, lesions in replicating DNA frequently cause a stalling of the replication machinery, as most DNA polymerases cannot cope with defective templates. This situation is aggravated by the fact that strand separation in preparation for DNA synthesis prevents common repair mechanisms relying on strand complementarity, such as base and nucleotide excision repair, from working properly. On the other hand, the replication process itself subjects the DNA to a series of hazardous transformations, ranging from the exposure of single-stranded DNA to topological contortions and the generation of nicks and fragments, which all bear the risk of inducing genomic instability. Dealing with these problems requires rapid and flexible responses, for which posttranslational protein modifications that act independently of protein synthesis are particularly well suited. Hence, it is not surprising that members of the ubiquitin family, particularly ubiquitin itself and SUMO, feature prominently in controlling many of the defensive and restorative measures involved in the protection of DNA during replication. In this review we will discuss the contributions of ubiquitin and SUMO to genome maintenance specifically as they relate to DNA replication. We will consider cases where the modifiers act during regular, i.e. unperturbed stages of replication, such as initiation, fork progression and termination, but also give an account of their functions in dealing with lesions, replication stalling and fork collapse.

Polymerase‐blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single‐stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re‐priming downstream of lesions can give rise to daughter‐strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9‐dependent mechanism of damage signaling is distinct from the Mrc1‐dependent, fork‐associated response to replication stress induced by conditions such as nucleotide depletion or replisome‐inherent problems, but reminiscent of replication‐independent checkpoint activation by single‐stranded DNA. Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase‐blocking lesions mainly emanates from Exo1‐processed, postreplicative daughter‐strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome‐ versus template‐induced checkpoint signaling.

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 depend 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 K_m 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.

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. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, 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.