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.

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.

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: insights from DNA complex structures.

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

A prominent ß-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation.

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1(49-616)) shows a prominent ß-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the ß-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the ß-hairpin is required for dimer formation in RECQ1(49-616) and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1(49-616) dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein-protein contacts are required for tetramer formation, one involves the ß-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.

An integrated approach for coastal cliff susceptibility: The case study of Procida Island (southern Italy).

Procida Island, located in the Gulf of Naples (southern Italy), is characterized by steep cliffed coasts, articulated in a succession of headlands and small embayments with narrow pocket beaches, such as Ciraccio and Chiaia, often characterized by instability. In this study, a methodology for coastal cliff susceptibility assessment has been conceived based on hydraulic and geomorphological characteristics, which supported the construction of a Cliff Stability Index (CSI). The geomorphological characteristics are related to the whole cliff face, the cliff material resistance, and the cliff failure mechanisms. The hydraulic actions on the cliff are related to the wave impact which is exerted by the breaking waves once the wave run-up distance exceeds the beach width. The index takes into account the slope of the cliff, the rock strength, the wave energy at the cliff base produced by the broken wave and the presence of defence structures at the cliff base. The resulting index classification, obtained by addition of the partial sub-indices, has been compared with the observed coastal cliff evolution from 1954 to 2021.

The human RECQ1 helicase is highly expressed in glioblastoma and plays an important role in tumor cell proliferation.

BACKGROUND: RecQ helicases play an essential role in the maintenance of genome stability. In humans, loss of RecQ helicase function is linked with predisposition to cancer and/or premature ageing. Current data show that the specific depletion of the human RECQ1 helicase leads to mitotic catastrophe in cancer cells and inhibition of tumor growth in mice. RESULTS: Here, we show that RECQ1 is highly expressed in various types of solid tumors. However, only in the case of brain gliomas, the high expression of RECQ1 in glioblastoma tissues is paralleled by a lower expression in the control samples due to the poor expression of RECQ1 in non-dividing tissues. This conclusion is validated by immunohistochemical analysis of a tissue microarray containing 63 primary glioblastomas and 19 perilesional tissue samples, as control. We also show that acute depletion of RECQ1 by RNAi results in a significant reduction of cellular proliferation, perturbation of S-phase progression, and spontaneous γ-H2AX foci formation in T98G and U-87 glioblastoma cells. Moreover, RECQ1 depleted T98G and U-87 cells are hypersensitive to HU or temozolomide treatment. CONCLUSIONS: Collectively, these results indicate that RECQ1 has a unique and important role in the maintenance of genome integrity. Our results also suggest that RECQ1 might represent a new suitable target for anti cancer therapies aimed to arrest cell proliferation in brain gliomas.

Sequential role of RAD51 paralog complexes in replication fork remodeling and restart.

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.

Gait pattern in Duchenne muscular dystrophy.

We investigated the gait pattern of 21 patients with Duchenne muscular dystrophy (DMD), compared to 10 healthy controls through 3D Gait Analysis. An overall observation of gait pattern in our DMD patients when compared to controls confirmed the data previously reported for small dystrophic groups. An excessive anterior tilt of pelvis and abnormal knee pattern in loading response phase were found. Since during the swing phase the DMD foot is too plantarflexed, patients adopt a higher flexion and abduction of the hip in order to advance the swinging limb. Velocity and cadence of DMD patients resulted similar to those calculated for healthy subjects, whereas stride length was reduced and step width was increased. We then divided the DMD patients in to two subgroups (treated with steroids and untreated), and we observed that the only statistically significant differences between the two groups in Gait Analysis parameters were found for the maximum of ankle power. 3D Gait Analysis gives objective and quantitative information about the gait pattern and the deviations due to muscular situation of DMD subjects; being our study a single moment evaluation, it is otherwise unable to unravel changes only detectable through serial analysis during the time course of the disease and, if any, due to the treatment.

XLF and H2AX function in series to promote replication fork stability.

XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7-dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax-/-:Xlf-/- cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

ATR-Mediated Global Fork Slowing and Reversal Assist Fork Traverse and Prevent Chromosomal Breakage at DNA Interstrand Cross-Links.

Interstrand cross-links (ICLs) are toxic DNA lesions interfering with DNA metabolism that are induced by widely used anticancer drugs. They have long been considered absolute roadblocks for replication forks, implicating complex DNA repair processes at stalled or converging replication forks. Recent evidence challenged this view, proposing that single forks traverse ICLs by yet elusive mechanisms. Combining ICL immunolabeling and single-molecule approaches in human cells, we now show that ICL induction leads to global replication fork slowing, involving forks not directly challenged by ICLs. Active fork slowing is linked to rapid recruitment of RAD51 to replicating chromatin and to RAD51/ZRANB3-mediated fork reversal. This global modulation of fork speed and architecture requires ATR activation, promotes single-fork ICL traverse-here, directly visualized by electron microscopy-and prevents chromosomal breakage by untimely ICL processing. We propose that global fork slowing by remodeling provides more time for template repair and promotes bypass of residual lesions, limiting fork-associated processing.

Are patients with hereditary spastic paraplegia different from patients with spastic diplegia during walking? Gait evaluation using 3D gait analysis.

Patients with hereditary spastic paraplegia (HSP) often resemble patients with mild spastic diplegia (SD), although their motor limitations differ. The aim of this study was to analyse quantitatively the gait of HSP and SD subjects in order to define the gait pattern in HSP and the differences between the two conditions. Fifteen subjects with HSP, 40 patients with SD and 20 healthy subjects underwent gait analysis (GA). The spatio-temporal and kinematic parameters at the proximal joints were found to be similar in HSP and SD, whereas the most significant differences were found at the knee and ankle joints. Both groups displayed a tendency for knee hyperextension in the midstance phase, but the duration of this hyperextension was longer in the HSP patients. This study shows that GA complements traditional clinical evaluations, making it possible to distinguish, clearly, between motor ability in HSP and in SD patients; the duration of the knee hyperextension during midstance was found to discriminate between the two gait patterns.

FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2.

Interstrand cross-link (ICL) hypersensitivity is a characteristic trait of Fanconi anemia (FA). Although FANCD2-associated nuclease 1 (FAN1) contributes to ICL repair, FAN1 mutations predispose to karyomegalic interstitial nephritis (KIN) and cancer rather than to FA. Thus, the biological role of FAN1 remains unclear. Because fork stalling in FAN1-deficient cells causes chromosomal instability, we reasoned that the key function of FAN1 might lie in the processing of halted replication forks. Here, we show that FAN1 contains a previously-uncharacterized PCNA interacting peptide (PIP) motif that, together with its ubiquitin-binding zinc finger (UBZ) domain, helps recruit FAN1 to ubiquitylated PCNA accumulated at stalled forks. This prevents replication fork collapse and controls their progression. Furthermore, we show that FAN1 preserves replication fork integrity by a mechanism that is distinct from BRCA2-dependent homologous recombination. Thus, targeting FAN1 activities and its interaction with ubiquitylated PCNA may offer therapeutic opportunities for treatment of BRCA-deficient tumors.

Replication fork reversal triggers fork degradation in BRCA2-defective cells.

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.BRCA2 is involved in both homologous recombination (HR) and the protection of stalled replication forks from degradation. Here the authors reveal how HR factors cooperate in fork remodeling, showing that BRCA2 supports RAD51 loading on the regressed arms of reversed replication forks to protect them from degradation.

Author Correction: FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2.

The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: This study was in part supported by the Swiss National Foundation Grant No.: 31003A-156023 to Alessandro Sartori.

Replication stress: getting back on track.

The replication-stress response enables the DNA replication machinery to overcome DNA lesions or intrinsic replication-fork obstacles, and it is essential to ensure faithful transmission of genetic information to daughter cells. Multiple replication stress­response pathways have been identified in recent years, thus raising questions about the specific and possibly redundant functions of these pathways. Here, we review the emerging mechanisms of the replication-stress response in mammalian cells and consider how they may influence the dynamics of the core DNA replication complex.

Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.

Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy.

DNA2 drives processing and restart of reversed replication forks in human cells.

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5'-to-3' polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.

Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition.

Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.