Replication Fork Research Articles

Targeting Replication Fork Processing Synergizes with PARP Inhibition to Potentiate Lethality in Homologous Recombination Proficient Ovarian Cancers.

Synthetic lethality in homologous recombination (HR)-deficient cancers caused by Poly (ADP-ribose) polymerase inhibitors (PARPi) has been classically attributed to its role in DNA repair. The mode of action of PARPi and resistance thereof are now believed to be predominantly replication associated. Therefore, effective combinatorial approaches of targeting replication fork processing along with HR-downregulation to target HR-proficient and possibly PARPi-resistant tumors are warranted. Stilbenes are a privileged class of molecules, which includeresveratrol, pterostilbene, piceatannol,etc,that modulate bothreplication processesand RAD51-expression. In this investigation, by screening asmalllibrary of stilbenes, including in-house synthesized molecules, trans-4,4'-dihydroxystilbene (DHS) was discovered as a potent natural agent, which downregulates RAD51 expression and HR repair (GFP-reporter assay). DHS induces extensive synergistic cell death in ovarian cancers when combined with talazoparib (PARPi). Mechanistically, DHS elicits replication-stress through severely impeding replication fork progress, speed, and inducing fork-asymmetry. This leads to robust induction of single stranded DNA (ssDNA) gaps and poly-ADP-ribosylation (PARylation) in S-phase cells, signifying issues related to lagging(Okazaki)strand synthesis. PARPi, which abrogates PARylation, potentiates DHS induced ssDNA gaps, and their conversion into lethal double strand breaks through MRE11 action. Furthermore, the combination is highly effective in mitigating ovarian tumor xenograft growth in SCID mice and exhibited a good therapeutic-index with no/minimal tissue-toxicity.

Control of Replication Stress Response by Cytosolic Fe-S Cluster Assembly (CIA) Machinery

Accurate DNA replication is essential for the maintenance of genome stability and the generation of healthy offspring. When DNA replication is challenged, signals accumulate at blocked replication forks that elicit a multifaceted cellular response, orchestrating DNA replication, DNA repair and cell cycle progression. This replication stress response promotes the recovery of DNA replication, maintaining chromosome integrity and preventing mutations. Defects in this response are linked to heightened genetic instability, which contributes to tumorigenesis and genetic disorders. Iron–sulfur (Fe-S) clusters are emerging as important cofactors in supporting the response to replication stress. These clusters are assembled and delivered to target proteins that function in the cytosol and nucleus via the conserved cytosolic Fe-S cluster assembly (CIA) machinery and the CIA targeting complex. This review summarizes recent advances in understanding the structure and function of the CIA machinery in yeast and mammals, emphasizing the critical role of Fe-S clusters in the replication stress response.

Replication stress: an early key event in ochratoxin a genotoxicity?

Abstract Ochratoxin A (OTA), a widespread food contaminant and potent renal carcinogen in rodents, is weakly genotoxic in mammalian cells. The mechanisms underlying OTA-induced genetic damage are still poorly understood. In its recent risk assessment, the European food safety authority (EFSA) considered that the specific spectrum of mutations and chromosomal damage induced by OTA may derive from unresolved replication stress. The aim of the present work was to experimentally test the hypothesis that OTA interferes with DNA replication and to characterize the cellular response to OTA-mediated replication stress. Using the DNA fiber assay to study replication fork dynamics at single molecule resolution, a small but statistically significant global delay in replication fork progression was observed in human kidney (HK-2) cells exposed to OTA at ≥ 10 µM. OTA-mediated interference with DNA replication was confirmed by a concentration-related decrease in incorporation of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) into newly replicating DNA in HK-2 cells arrested in late G1/S via double thymidine block and treated with OTA during S phase. Western blot and immunofluorescence analyses revealed a significant concentration-related increase in γH2AX in cells exposed to OTA. Co-localization of γH2AX foci with 5-chloro-2′-deoxyuridine (CldU) incorporated into cells during S phase and increased γH2AX labeling along newly replicating chromatin fibers visualized using the extended chromatin fiber assay support a replication-coupled mechanism of OTA-induced DNA damage. Experiments with cells synchronized in late G1/S or late G2 demonstrated that exposure of cells to OTA during S phase, but not mitosis, leads to a significant concentration-related increase in H2AX, providing further evidence that OTA may act primarily during S phase of the cell cycle. However, OTA did not appear to efficiently activate ATR-Chk1 and ATM-Chk2 DNA damage response pathways, suggesting that cells with under-replicated DNA or unresolved DNA damage may escape checkpoint control and may continue into mitosis, with potentially deleterious consequences for genomic integrity. Overall, results from this study provide first experimental evidence for perturbation of the S phase replisome machinery by OTA and point toward replication stress as an early key event in OTA genotoxicity.

G-quadruplex-stalled eukaryotic replisome structure reveals helical inchworm DNA translocation.

DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork collisions with preformed G4s in the parental DNA using reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by arresting the CMG helicase. Cryo-electron microscopy structures of stalled yeast and human CMG complexes reveal that the folded G4 is lodged inside the central CMG channel, arresting translocation. The G4 stabilizes the CMG at distinct translocation intermediates, suggesting an unprecedented helical inchworm mechanism for DNA translocation. These findings illuminate the eukaryotic replication fork mechanism under normal and perturbed conditions.

Mutations and structural variants arising during double-strand break repair.

DNA synthesis during repair of a double-strand chromosome break by homologous recombination exhibits a high rate of mutation compared to normal replication. Using a genetic system in budding yeast, we isolated thousands of mutations occurring during repair. We conclude that the repair replication fork appears to have the two DNA strands open ∼80 bp ahead of the DNA polymerase, but the strands re-anneal rapidly behind the polymerase. Additionally, we analyzed interchromosomal template switching, in which the partially copied DNA strand dissociates and pairs with a new template at a short stretch of perfectly matching bases (microhomology), and resumes copying. We show that these apparent microhomology-mediated template switching events in fact require the pairing of ∼200 bp of imperfectly matching bases (homeology).

Chromatin assembly factor 1 subunit A promotes TLS pathway by recruiting E3 ubiquitin ligase RAD18 in cancer cells

The translesion DNA synthesis (TLS) pathway mediated by proliferating cell nuclear antigen (PCNA) monoubiquitination is an essential mechanism by which cancer cells bypass DNA damage caused by DNA damage to maintain genomic stability and cell survival. Chromatin assembly factor 1 subunit A (CHAF1A) traditionally promotes histone assembly during DNA replication. Here, we revealed that CHAF1A is a novel regulator of the TLS pathway in cancer cells. CHAF1A promotes restart and elongation of the replication fork under DNA replication stress. Mechanistically, the C-terminal domain of CHAF1A directly interacts with E3 ubiquitin ligase RAD18, enhancing RAD18 binding on the stalled replication fork. CHAF1A facilitates PCNA K164 monoubiquitination mediated by RAD18, thereby promoting the recruitment of Y-family DNA polymerases and enhancing cancer cell resistance to DNA damage. In addition, CHAF1A-mediated RAD18 recruitment and PCNA monoubiquitination are independent of the CHAF1A-PCNA interaction and its histone assembly function. Taken together, these findings improve our understanding of the mechanisms that regulate the TLS pathway and provide insights into the relationship between CHAF1A and DNA replication stress in cancer cells.

Quantitative Chromatin Protein Dynamics During Replication Origin Firing in Human Cells.

Quantitative Chromatin Protein Dynamics During Replication Origin Firing in Human Cells.

Emerging Role of Natural Topoisomerase Inhibitors as Anticancer agents.

Topoisomerases I and II are the functionally two forms of DNA topoisomerase. In anticancer research, novel anticancer chemotherapeutical capable of blocking topoisomerase enzymes have been discovered. Most commonly, topoisomerase causes replication fork arrest and doublestrand breaks, and this is how a clinically successful topoisomerase-targeting anticancer medicines work. Unfortunately, this novel mechanism of action has been linked to the development of secondary malignancies as well as cardiotoxicity. The specific binding locations and mechanisms of topoisomerase poisons have been identified by studying the structures of topoisomerase-drug-DNA ternary complexes. Recent breakthroughs in science have revealed that isoform-specific human topoisomerase II poison could be created as safer anticancer drug molecules. It may also be able to develop catalytic inhibitors of topoisomerases by focusing on their inactive conformations. In addition to this, the discovery of new bacterial topoisomerase inhibitor molecules and regulatory proteins could lead to the discovery of new human topoisomerase inhibitors. As a result, biologists, organic chemists, and medicinal chemists worldwide have been identifying, designing, synthesizing, and testing a variety of novel topoisomerase-targeting bioactive compounds. This review focused on topoisomerase inhibitors, their mechanisms of action, and different types of topoisomerase inhibitors that have been developed during the last ten years.

Discovery of new inhibitors of nuclease MRE11.

Discovery of new inhibitors of nuclease MRE11.

Beyond interacting with Rap1: Dissecting the roles of Rif1.

Beyond interacting with Rap1: Dissecting the roles of Rif1.

Increased levels of lagging strand polymerase α in an adult stem cell lineage affect replication-coupled histone incorporation.

Stem cells display asymmetric histone inheritance, while nonstem progenitor cells exhibit symmetric patterns in the Drosophila male germ line. Here, we report that components involved in lagging strand synthesis, DNA polymerases α and δ, have substantially reduced levels in stem cells compared to progenitor cells, and this promotes local asymmetry of parental histone incorporation at the replication fork. Compromising Polα genetically induces the local replication-coupled histone incorporation pattern in progenitor cells to resemble that in stem cells, seen by both nuclear localization patterns and chromatin fibers. This is recapitulated using a Polα inhibitor in a concentration-dependent manner. The local old versus new histone asymmetry is comparable between stem cells and progenitor cells at both S phase and M phase. Together, these results indicate that developmentally programmed expression of key DNA replication components is important to shape stem cell chromatin. Furthermore, manipulating one crucial DNA replication component can induce replication-coupled histone dynamics in nonstem cells to resemble those in stem cells.

Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol.

Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol.

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS

Processivity clamps mediate polymerase switching for translesion synthesis (TLS). All three Escherichia coli TLS polymerases interact with the β2 processivity clamp through a conserved clamp-binding motif (CBM), which is indispensable for TLS. Notably, Pol IV also interacts weakly with the rim of the clamp through non-CBM residues. Ablating this "rim contact" in cells results in selective sensitivity to DNA-damaging agents, raising the question how the rim contact contributes to TLS. Here, we show that the rim contact is critical for TLS past a strong replication block but barely necessary for a weak blocking lesion. Within the in vitro reconstituted E. coli replisome, the rim mutation compromises Pol IV-mediated TLS past 3-deaza-methyl dA, a strong block, whereas barely affecting TLS past N2-furfuryl dG, a weak block. Similar observations are also made in E. coli cells bearing a single copy of these lesions in the genome. At lesion-stalled replication forks, single-stranded DNA binding protein locally enriches Pol IV, enabling it to bind the low-affinity rim site. This interaction poises Pol IV to better compete with Pol III, the replicative polymerase, which competitively inhibits Pol IV from interacting with the clamp through its CBM. We propose that this bipartite clamp interaction enables Pol IV to rapidly resolve lesion-stalled replication at a strong block through TLS, which reduces damage-induced mutagenesis.

BMP signaling promotes zebrafish heart regeneration via alleviation of replication stress

In contrast to mammals, adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly, we found that regenerating cardiomyocytes experience DNA replication stress, which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes, human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication.

DNA replication dynamics are associated with genome composition in Plasmodium species.

Plasmodium species have variable genome compositions: many have an A/Tcontent>80%, while others are similar in composition to human cells. Here, we made a direct comparison of DNA replication dynamics in two Plasmodium species whose genomes differ by ∼20% A/Tcontent. This yielded fundamental insights into how DNA composition may affect replication. The highly A/T-biased genome of Plasmodium falciparum showed unusual replication dynamics that were not observed in the more balanced Plasmodium knowlesi-which had dynamics more like those of human cell lines. Replication forks moved 50% slower in P. falciparum than in P. knowlesi. In P. falciparum, replication forks slowed down over the course of S-phase, whereas in P. knowlesi, fork speed increased as in human cells. Furthermore, in both P. knowlesi and human cells, replication forks were strikingly slowed by sequences of particularly high A/Tbias, but in P. falciparum, although replication forks were inherently slow, they were not particularly slow in such biased sequences. Thus, the replisome of P. falciparum may have evolved alongside its extremely biased genome, making it unusually robust to sequence bias. Since several antimalarial drugs act to stall DNA replication, this study may have implications for the effectiveness of, and development of, antimalarial therapies.

Ubiquitin protease Ubp1 cooperates with Ubp10 and Ubp12 to revert lysine-164 PCNA ubiquitylation at replication forks.

Proliferating cell nuclear antigen (PCNA) is essential for the faithful duplication of eukaryotic genomes. PCNA also orchestrates events necessary to address threats to genomic integrity, such as the DNA damage tolerance (DDT) response, a mechanism by which eukaryotic cells bypass replication-blocking lesions to maintain replisome stability. DDT is regulated by the ubiquitylation of PCNA and the consequent recruitment of specialized polymerases that ensure replication continuity. We have recently described that the deubiquitylases Ubp10 and Ubp12 modulate DDT events by reverting the ubiquitylation of PCNA in Saccharomyces cerevisiae. This study identifies Ubp1 as a novel PCNA deubiquitylase that cooperates with Ubp10 and Ubp12 in the regulation of DDT during DNA replication. Ubp1, previously known as a cytoplasmic protein, also localizes to the nucleus, where it associates with DNA replication forks. Additionally, Ubp1 interacts with and deubiquitylates PCNA. Here, we provide evidence that Ubp1 collaborates with Ubp10 and Ubp12 to facilitate DNA replication by efficiently reverting PCNAK164 ubiquitylation at replication forks under conditions free from exogenous perturbations. Consequently, the deletion of UBP1, UBP10, and UBP12 leads to persistent ubiquitylation of PCNAK164 and a marked delay in S phase progression.

Overexpression of the NEK8 kinase inhibits homologous recombination.

Homologous recombination maintains genome stability by repairing double strand breaks and protecting replication fork stability. Defects in homologous recombination results in cancer predisposition but can be exploited due to increased sensitivity to certain chemotherapeutics such as PARP inhibitors. The NEK8 kinase has roles in the replication response and homologous recombination. NEK8 is overexpressed in breast cancer, but the impact of NEK8 overexpression on homologous recombination has not been determined. Here, we demonstrate NEK8 overexpression inhibits RAD51 focus formation resulting in a defect in homologous recombination and degradation of stalled replication forks. Importantly, NEK8 overexpression sensitizes cells to the PARP inhibitor, Olaparib. Together, our results suggest NEK8 overexpressing tumors may be recombination-deficient and respond to chemotherapeutics that target defects in recombination such as Olaparib.

Transcription-replication conflicts drive R-loop-dependent nucleosome eviction and require DOT1L activity for transcription recovery.

Progressing transcription and replication machineries profoundly impact their underlying chromatin template. Consequently, transcription-replication conflict (TRC) sites are vulnerable to chromatin and epigenome alterations, provoking genome instability. Here, we engineered an inducible TRC reporter system using a genome-integrated R-loop-prone sequence and characterized the dynamic changes of the local chromatin structure inflicted by TRCs, leading to reduced nucleosome occupancy and replication fork blockage. Strikingly, inducing a small number of TRCs on the genome results in a measurable global replication stress response. Furthermore, we find a TRC-dependent increase in H3K79 methylation specifically at the R-loop forming TRC site. Accordingly, inhibition of the H3K79 methyltransferase DOT1L leads to reduced transcriptional output and an exacerbated DNA damage response, suggesting that deposition of this mark is required for effective transcription recovery and resolution of TRCs. Our work shows the molecular dynamics and reveals a specific epigenetic modifier bookmarking TRC sites, relevant to cancer and other diseases.

Replication-IDentifier links epigenetic and metabolic pathways to the replication stress response

Perturbation of DNA replication, for instance by hydroxyurea-dependent dNTP exhaustion, often leads to stalling or collapse of replication forks. This triggers a replication stress response that stabilizes these forks, activates cell cycle checkpoints, and induces expression of DNA damage response genes. While several factors are known to act in this response, the full repertoire of proteins involved remains largely elusive. Here, we develop Replication-IDentifier (Repli-ID), which allows for genome-wide identification of regulators of DNA replication in Saccharomyces cerevisiae. During Repli-ID, the replicative polymerase epsilon (Pol ε) is tracked at a barcoded origin of replication by chromatin immunoprecipitation (ChIP) coupled to next-generation sequencing of the barcode in thousands of hydroxyurea-treated yeast mutants. Using this approach, 423 genes that promote Pol ε binding at replication forks were uncovered, including LGE1 and ROX1. Mechanistically, we show that Lge1 affects replication initiation and/or fork stability by promoting Bre1-dependent H2B mono-ubiquitylation. Rox1 affects replication fork progression by regulating S-phase entry and checkpoint activation, hinging on cellular ceramide levels via transcriptional repression of SUR2. Thus, Repli-ID provides a unique resource for the identification and further characterization of factors and pathways involved in the cellular response to DNA replication perturbation.

Transcription Factor Inhibition as a Potential Additional Mechanism of Action of Pyrrolobenzodiazepine (PBD) Dimers

Background: The pyrrolobenzodiazepine (PBD) dimer SJG-136 reached Phase II clinical trials in ovarian cancer and leukaemia in the UK and USA in the 2000s. Several structural analogues of SJG-136 are currently in clinical development as payloads for Antibody-Drug Conjugates (ADCs). There is growing evidence that PBDs exert their pharmacological effects through inhibition of transcription factors (TFs) in addition to arrest at the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. Hence, PBDs can be used to target specific DNA sequences to inhibit TFs as a novel anticancer therapy. Objective: To explore the ability of SJG-136 to bind to the cognate sequences of transcription factors using a previously described HPLC/MS method, to obtain preliminary mechanistic evidence of its ability to inhibit transcription factors (TF), and to determine its effect on TF-dependent gene expression. Methods: An HPLC/MS method was used to assess the kinetics and thermodynamics of adduct formation between the PBD dimer SJG-136 and the cognate recognition sequence of the TFs NF-κB, EGR-1, AP-1, and STAT3. CD spectroscopy, molecular dynamics simulations, and gene expression analyses were used to rationalize the findings of the HPLC/MS study. Results: Notable differences in the rate and extent of adduct formation were observed with different DNA sequences, which might explain the variations in cytotoxicity of SJG-136 observed across different tumour cell lines. The differences in adduct formation result in variable downregulation of several STAT3-dependent genes in the human colon carcinoma cell line HT-29 and the human breast cancer cell line MDA-MB-231. Conclusions: SJG-136 can disrupt transcription factor-mediated gene expression, which contributes to its exceptional cytotoxicity in addition to the DNA-strand cleavage initiated by its ability to crosslink DNA.