Werner syndrome (WS) is an autosomal recessive disease caused by loss of function of WRN. WS is a segmental progeroid disease and shows early onset or increased frequency of many characteristics of normal aging. WRN possesses helicase, annealing, strand exchange, and exonuclease activities and acts on a variety of DNA substrates, even complex replication and recombination intermediates. Here, we review the genetics, biochemistry, and probably physiological functions of the WRN protein. Although its precise role is unclear, evidence suggests WRN plays a role in pathways that respond to replication stress and maintain genome stability particularly in telomeric regions.

Homologous recombination (HR) plays an essential role in the repair of DNA double-strand breaks (DSBs), replication stress responses, and genome maintenance. However, unregulated HR during replication can impair genome duplication and compromise genome stability. The mechanisms underlying HR regulation during DNA replication are obscure. Here, we find that RTEL1 helicase, RAD51, and RAD51 paralogs are enriched at stalled replication sites. The absence of RTEL1 leads to an increase in the RAD51-mediated HR and fork reversal during replication and affects genome-wide replication, which can be rescued by co-depleting RAD51 and RAD51 paralogs. Interestingly, co-depletion of fork remodelers such as SMARCAL1/ZRANB3/HLTF/FBH1 and expression of HR-defective RAD51 mutants also rescues replication defects in RTEL1-deficient cells. The anti-recombinase function of RTEL1 during replication depends on its interaction with PCNA and helicase activity. Together, our data identify the role of RTEL1 helicase in restricting RAD51-mediated fork reversal and HR activity to facilitate error-free genome duplication.

Fanconi anemia (FA) is a hereditary disorder characterized by a deficiency in the repair of DNA interstrand crosslinks and the response to replication stress. Endogenous DNA damage, most likely caused by aldehydes, severely affects hematopoietic stem cells in FA, resulting in progressive bone marrow failure and the development of leukemia. Recent studies revealed that expression levels of SLFN11 affect the replication stress response and are a strong determinant in cell killing by DNA-damaging cancer chemotherapy. Because SLFN11 is highly expressed in the hematopoietic system, we speculated that SLFN11 may have a significant role in FA pathophysiology. Indeed, we found that DNA damage sensitivity in FA cells is significantly mitigated by the loss of SLFN11 expression. Mechanistically, we demonstrated that SLFN11 destabilizes the nascent DNA strands upon replication fork stalling. In this review, we summarize our work regarding an interplay between SLFN11 and the FA pathway, and the role of SLFN11 in the response to replication stress.

DNA replication is remarkably accurate with estimates of only a handful of mutations per human genome per cell division cycle. Replication stress caused by DNA lesions, transcription-replication conflicts, and other obstacles to the replication machinery must be efficiently overcome in ways that minimize errors and maximize completion of DNA synthesis. Replication fork reversal is one mechanism that helps cells tolerate replication stress. This process involves reannealing of parental template DNA strands and generation of a nascent-nascent DNA duplex. While fork reversal may be beneficial by facilitating DNA repair or template switching, it must be confined to the appropriate contexts to preserve genome stability. Many enzymes have been implicated in this process including ATP-dependent DNA translocases like SMARCAL1, ZRANB3, HLTF, and the helicase FBH1. In addition, the RAD51 recombinase is required. Many additional factors and regulatory activities also act to ensure reversal is beneficial instead of yielding undesirable outcomes. Finally, reversed forks must also be stabilized and often need to be restarted to complete DNA synthesis. Disruption or deregulation of fork reversal causes a variety of human diseases. In this review we will describe the latest models for reversal and key mechanisms of regulation.

DNA replication ensures the complete and accurate duplication of the genome. The traditional approach to analysing perturbation of DNA replication is to use chemical inhibitors, such as hydroxyurea or aphidicolin, that slow or stall replication fork progression throughout the genome. An alternative approach is to perturb replication at a single site in the genome that permits a more forensic investigation of the cellular response to the stalling or disruption of a replication fork. This has been achieved in several organisms using different systems that share the common feature of utilizing the high affinity binding of a protein to a defined DNA sequence that is integrated into a specific locus in the host genome. Protein-mediated replication fork blocking systems of this sort have proven very valuable in defining how cells cope with encountering a barrier to fork progression. In this review, we compare protein-based replication fork barrier systems from different organisms that have been developed to generate site-specific replication fork perturbation.

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.

Enzymes of the apolipoprotein B mRNA editing catalytic polypeptide like (APOBEC) family are cytosine deaminases that convert cytosine to uracil in DNA and RNA. Among these proteins, APOBEC3 sub-family members, APOBEC3A (A3A) and APOBEC3B (A3B), are prominent sources of mutagenesis in cancer cells. The aberrant expression of A3A and A3B in cancer cells leads to accumulation of mutations with specific single-base substitution (SBS) signatures, characterized by C→T and C→G changes, in a number of tumor types. In addition to fueling mutagenesis, A3A and A3B, particularly A3A, induce DNA replication stress, DNA damage, and chromosomal instability through their catalytic activities, triggering a range of cellular responses. Thus, A3A/B have emerged as key drivers of genome evolution during cancer development, contributing to tumorigenesis, tumor heterogeneity, and therapeutic resistance. Yet, the expression of A3A/B in cancer cells presents a cancer vulnerability that can be exploited therapeutically. In this review, we discuss the recent studies that shed light on the mechanisms regulating A3A expression and the impact of A3A in cancer. We also review recent advances in the development of A3A inhibitors and provide perspectives on the future directions of A3A research.

Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/- ) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.