The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.

Complete and accurate DNA replication requires the progression of replication forks through DNA damage, actively transcribed regions, structured DNA and compact chromatin. Recent studies have revealed a remarkable plasticity of the replication process in dealing with these obstacles, which includes modulation of replication origin firing, of the architecture of replication forks, and of the functional organization of the replication machinery in response to replication stress. However, these specialized mechanisms also expose cells to potentially dangerous transactions while replicating DNA. In this Review, we discuss how replication forks are actively stalled, remodelled, processed, protected and restarted in response to specific types of stress. We also discuss adaptations of the replication machinery and the role of chromatin modifications during these transactions. Finally, we discuss interesting recent data on the relevance of replication fork plasticity to human health, covering its role in tumorigenesis, its crosstalk with innate immunity responses and its potential as an effective cancer therapy target.

DNA-PKcs promotes fork reversal and chemoresistance.

The DNA-PKcs kinase mediates the repair of DNA double-strand breaks via classical non-homologous end joining (NHEJ). DNA-PKcs is also recruited to active replication forks, although a role for DNA-PKcs in the control of fork dynamics is unclear. Here, we identify a crucial role for DNA-PKcs in promoting fork reversal, a process that stabilizes stressed replication forks and protects genome integrity. DNA-PKcs promotes fork reversal and slowing in response to several replication stress-inducing agents in a manner independent of its role in NHEJ. Cells lacking DNA-PKcs activity show increased DNA damage during S-phase and cellular sensitivity to replication stress. Notably, prevention of fork slowing and reversal via DNA-PKcs inhibition efficiently restores chemotherapy sensitivity in BRCA2-deficient mammary tumors with acquired PARPi resistance. Together, our data uncover a new key regulator of fork reversal and show how DNA-PKcs signaling can be manipulated to alter fork dynamics and drug resistance in cancer.

Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.

RAD51 restricts DNA over-replication from re-activated origins.

Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.

HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis.

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

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.

Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient ß-catenin/Hoxa9/Prmt1 axis.

ABSTRACT: Maintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, ß-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although ß-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, ß-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of ß-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient ß-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase.

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.

DNA replication and replication stress response in the context of nuclear architecture.

The DNA replication process needs to be coordinated with other DNA metabolism transactions and must eventually extend to the full genome, regardless of chromatin status, gene expression, secondary structures and DNA lesions. Completeness and accuracy of DNA replication are crucial to maintain genome integrity, limiting transformation in normal cells and offering targeting opportunities for proliferating cancer cells. DNA replication is thus tightly coordinated with chromatin dynamics and 3D genome architecture, and we are only beginning to understand the underlying molecular mechanisms. While much has recently been discovered on how DNA replication initiation is organised and modulated in different genomic regions and nuclear territories-the so-called "DNA replication program"-we know much less on how the elongation of ongoing replication forks and particularly the response to replication obstacles is affected by the local nuclear organisation. Also, it is still elusive how specific components of nuclear architecture participate in the replication stress response. Here, we review known mechanisms and factors orchestrating replication initiation, and replication fork progression upon stress, focusing on recent evidence linking genome organisation and nuclear architecture with the cellular responses to replication interference, and highlighting open questions and future challenges to explore this exciting new avenue of research.

PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity.

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occur during both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency.

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.

Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.

TARG1 protects against toxic DNA ADP-ribosylation.

ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed 'DarT' from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation.

CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage.

The CDC7 kinase is essential for the activation of DNA replication origins and has been implicated in the replication stress response. Using a highly specific chemical inhibitor and a chemical genetic approach, we now show that CDC7 activity is required to coordinate multiple MRE11-dependent processes occurring at replication forks, independently from its role in origin firing. CDC7 localizes at replication forks and, similarly to MRE11, mediates active slowing of fork progression upon mild topoisomerase inhibition. Both proteins are also retained on stalled forks, where they promote fork processing and restart. Moreover, MRE11 phosphorylation and localization at replication factories are progressively lost upon CDC7 inhibition. Finally, CDC7 activity at reversed forks is required for their pathological MRE11-dependent degradation in BRCA2-deficient cells. Thus, upon replication interference CDC7 is a key regulator of fork progression, processing and integrity. These results highlight a dual role for CDC7 in replication, modulating both initiation and elongation steps of DNA synthesis, and identify a key intervention point for anticancer therapies exploiting replication interference.

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.

The MMS22L-TONSL heterodimer directly promotes RAD51-dependent recombination upon replication stress.

Homologous recombination (HR) is a key pathway that repairs DNA double-strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L-TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L-TONSL associates with replication protein A (RPA)-coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR-mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L-TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51-ssDNA nucleoprotein filament formation and RAD51-dependent strand exchange activity in vitro Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L-TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR-mediated restart in vivo.