Replication fork reversal in eukaryotes: from dead end to dynamic response.

The remodelling of replication forks into four-way junctions following replication perturbation, known as fork reversal, was hypothesized to promote DNA damage tolerance and repair during replication. Albeit conceptually attractive, for a long time fork reversal in vivo was found only in prokaryotes and specific yeast mutants, calling its evolutionary conservation and physiological relevance into question. Based on the recent visualization of replication forks in metazoans, fork reversal has emerged as a global, reversible and regulated process, with intriguing implications for replication completion, chromosome integrity and the DNA damage response. The study of the putative in vivo roles of recently identified eukaryotic factors in fork remodelling promises to shed new light on mechanisms of genome maintenance and to provide novel attractive targets for cancer therapy.

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template.

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.

Carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double-strand breaks and a DNA damage response in its host cells.

The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pylori-associated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigen-binding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors--p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)--and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.

Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose).

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.

FBH1 Catalyzes Regression of Stalled Replication Forks.

DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression of a model replication fork in vitro and promotes fork regression in vivo in response to replication perturbation. Cells respond to fork stalling by activating checkpoint responses requiring signaling through stress-activated protein kinases. Importantly, we show that FBH1, through its helicase activity, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose a model whereby FBH1 promotes early checkpoint signaling by remodeling of stalled DNA replication forks.

Visualization and interpretation of eukaryotic DNA replication intermediates in vivo by electron microscopy.

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of genomic DNA, extracted from budding yeast, Xenopus egg extracts, or cultured mammalian cells.

New histone supply regulates replication fork speed and PCNA unloading.

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.

Mutation frequency dynamics in HPRT locus in culture-adapted human embryonic stem cells and induced pluripotent stem cells correspond to their differentiated counterparts.

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates.

Oncogene-induced DNA replication stress activates the DNA damage response (DDR), a crucial anticancer barrier. DDR inactivation in these conditions promotes genome instability and tumor progression, but the underlying molecular mechanisms are elusive. We found that overexpression of both Cyclin E and Cdc25A rapidly slowed down replication forks and induced fork reversal, suggestive of increased topological stress. Surprisingly, these phenotypes, per se, are neither associated with chromosomal breakage nor with significant DDR activation. Oncogene-induced DNA breakage and DDR activation instead occurred upon persistent G2/M arrest or, in a checkpoint-defective context, upon premature CDK1 activation. Depletion of MUS81, a cell cycle-regulated nuclease, markedly limited chromosomal breakage and led to further accumulation of reversed forks. We propose that nucleolytic processing of unusual replication intermediates mediates oncogene-induced genotoxicity and that limiting such processing to mitosis is a central anti-tumorigenic function of the DNA damage checkpoints.

Topoisomerase I poisoning results in PARP-mediated replication fork reversal.

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics.