Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

RNase E searches for cleavage sites in RNA by linear diffusion: direct evidence from single-molecule FRET.

The ability of obstacles in cellular transcripts to protect downstream but not upstream sites en masse from attack by RNase E has prompted the hypothesis that this mRNA-degrading endonuclease may scan 5'-monophosphorylated RNA linearly for cleavage sites, starting at the 5' end. However, despite its proposed regulatory importance, the migration of RNase E on RNA has never been directly observed. We have now used single-molecule FRET to monitor the dynamics of this homotetrameric enzyme on RNA. Our findings reveal that RNase E slides along unpaired regions of RNA without consuming a molecular source of energy such as ATP and that its forward progress can be impeded when it encounters a large structural discontinuity. This movement, which is bidirectional, occurs in discrete steps of variable length and requires an RNA ligand much longer than needed to occupy a single RNase E subunit. These results indicate that RNase E scans for cleavage sites by one-dimensional diffusion and suggest a possible molecular mechanism.

Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts.

PARP1&2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1&2 at DNA lesions. Here, we report that unlike Parp2 -/- mice, which develop normally, mice expressing catalytically-inactive Parp2 (E534A, Parp2 EA/EA ) succumb to Tp53- and Chk2 -dependent erythropoietic failure in utero , mirroring Lig1 -/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5'-phosphorylated nicks (5'p-nicks) between Okazaki fragments, typically resolved by Lig1. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, particularly harmful to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5'p-nicks explains the detrimental effects of PARP2 inhibition on erythropoiesis, revealing the mechanism behind the PARPi-induced anemia and leukemia, especially those with TP53/CHK2 loss. Significance: This work shows that the hematological toxicities associated with PARP inhibitors stem not from impaired PARP1 or PARP2 enzymatic activity but rather from the presence of inactive PARP2 protein. Mechanistically, these toxicities reflect a unique role of PARP2 at 5'-phosphorylated DNA nicks during DNA replication in erythroblasts.

Stepwise requirements for polymerases δ and θ in theta-mediated end joining.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells.

DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells.

BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair.

Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic; however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele, whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

A two-step mechanism governing PARP1-DNA retention by PARP inhibitors.

PARP inhibitors (PARPi) have emerged as promising cancer therapeutics capable of targeting specific DNA repair pathways, but their mechanism of action with respect to PARP1-DNA retention remains unclear. Here, we developed single-molecule assays to directly monitor the retention of PARP1 on DNA lesions in real time. Our study reveals a two-step mechanism by which PARPi modulate the retention of PARP1 on DNA lesions, consisting of a primary step of catalytic inhibition via binding competition with NAD+ followed by an allosteric modulation of bound PARPi. While clinically relevant PARPi exhibit distinct allosteric modulation activities that can either increase retention of PARP1 on DNA or induce its release, their retention potencies are predominantly determined by their ability to outcompete NAD+ binding. These findings provide a mechanistic basis for improved PARPi selection according to their characteristic activities and enable further development of more potent inhibitors.

Loss of Nuclear Envelope Integrity and Increased Oxidant Production Cause DNA Damage in Adult Hearts Deficient in PKP2: A Molecular Substrate of ARVC.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by high propensity to life-threatening arrhythmias and progressive loss of heart muscle. More than 40% of reported genetic variants linked to ARVC reside in the PKP2 gene, which encodes the PKP2 protein (plakophilin-2). METHODS: We describe a comprehensive characterization of the ARVC molecular landscape as determined by high-resolution mass spectrometry, RNA sequencing, and transmission electron microscopy of right ventricular biopsy samples obtained from patients with ARVC with PKP2 mutations and left ventricular ejection fraction >45%. Samples from healthy relatives served as controls. The observations led to experimental work using multiple imaging and biochemical techniques in mice with a cardiac-specific deletion of Pkp2 studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: Samples from patients with ARVC present a loss of nuclear envelope integrity, molecular signatures indicative of increased DNA damage, and a deficit in transcripts coding for proteins in the electron transport chain. Mice with a cardiac-specific deletion of Pkp2 also present a loss of nuclear envelope integrity, which leads to DNA damage and subsequent excess oxidant production (O2.- and H2O2), the latter increased further under mechanical stress (isoproterenol or exercise). Increased oxidant production and DNA damage is recapitulated in human induced pluripotent stem cell-derived PKP2-deficient myocytes. Furthermore, PKP2-deficient cells release H2O2 into the extracellular environment, causing DNA damage and increased oxidant production in neighboring myocytes in a paracrine manner. Treatment with honokiol increases SIRT3 (mitochondrial nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-3) activity, reduces oxidant levels and DNA damage in vitro and in vivo, reduces collagen abundance in the right ventricular free wall, and has a protective effect on right ventricular function. CONCLUSIONS: Loss of nuclear envelope integrity and subsequent DNA damage is a key substrate in the molecular pathology of ARVC. We show transcriptional downregulation of proteins of the electron transcript chain as an early event in the molecular pathophysiology of the disease (before loss of left ventricular ejection fraction <45%), which associates with increased oxidant production (O2.- and H2O2). We propose therapies that limit oxidant formation as a possible intervention to restrict DNA damage in ARVC.

USP1-trapping lesions as a source of DNA replication stress and genomic instability.

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.

A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination.

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.

Super-resolution visualization of distinct stalled and broken replication fork structures.

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). Super-resolution microscopy has made it possible to directly observe replication stress and DNA damage inside cells, however new approaches to sample preparation and analysis are required. Here we develop and apply multicolor single molecule microscopy to visualize individual replication forks under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We observe RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. By implementing novel super-resolution imaging assays, we are thus able to discern closely related replication fork stress motifs and their repair pathways.

The molecular basis and disease relevance of non-homologous DNA end joining.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments.

Numerous anti-cancer drugs perturb thymidylate biosynthesis and lead to genomic uracil incorporation contributing to their antiproliferative effect. Still, it is not yet characterized if uracil incorporations have any positional preference. Here, we aimed to uncover genome-wide alterations in uracil pattern upon drug treatments in human cancer cell line models derived from HCT116. We developed a straightforward U-DNA sequencing method (U-DNA-Seq) that was combined with in situ super-resolution imaging. Using a novel robust analysis pipeline, we found broad regions with elevated probability of uracil occurrence both in treated and non-treated cells. Correlation with chromatin markers and other genomic features shows that non-treated cells possess uracil in the late replicating constitutive heterochromatic regions, while drug treatment induced a shift of incorporated uracil towards segments that are normally more active/functional. Data were corroborated by colocalization studies via dSTORM microscopy. This approach can be applied to study the dynamic spatio-temporal nature of genomic uracil.

Cancer-associated mutations in the iron-sulfur domain of FANCJ affect G-quadruplex metabolism.

FANCJ/BRIP1 is an iron-sulfur (FeS) cluster-binding DNA helicase involved in DNA inter-strand cross-link (ICL) repair and G-quadruplex (G4) metabolism. Mutations in FANCJ are associated with Fanconi anemia and an increased risk for developing breast and ovarian cancer. Several cancer-associated mutations are located in the FeS domain of FANCJ, but how they affect FeS cluster binding and/or FANCJ activity has remained mostly unclear. Here we show that the FeS cluster is indispensable for FANCJ's ability to unwind DNA substrates in vitro and to provide cellular resistance to agents that induce ICLs. Moreover, we find that FANCJ requires an intact FeS cluster for its ability to unfold G4 structures on the DNA template in a primer extension assay with the lagging-strand DNA polymerase delta. Surprisingly, however, FANCJ variants that are unable to bind an FeS cluster and to unwind DNA in vitro can partially suppress the formation of replisome-associated G4 structures that we observe in a FANCJ knock-out cell line. This may suggest a partially retained cellular activity of FANCJ variants with alterations in the FeS domain. On the other hand, FANCJ knock-out cells expressing FeS cluster-deficient variants display a similar-enhanced-sensitivity towards pyridostatin (PDS) and CX-5461, two agents that stabilise G4 structures, as FANCJ knock-out cells. Mutations in FANCJ that abolish FeS cluster binding may hence be predictive of an increased cellular sensitivity towards G4-stabilising agents.

Preserving genome integrity in human cells via DNA double-strand break repair.

The efficient maintenance of genome integrity in the face of cellular stress is vital to protect against human diseases such as cancer. DNA replication, chromatin dynamics, cellular signaling, nuclear architecture, cell cycle checkpoints, and other cellular activities contribute to the delicate spatiotemporal control that cells utilize to regulate and maintain genome stability. This perspective will highlight DNA double-strand break (DSB) repair pathways in human cells, how DNA repair failures can lead to human disease, and how PARP inhibitors have emerged as a novel clinical therapy to treat homologous recombination-deficient tumors. We briefly discuss how failures in DNA repair produce a permissive genetic environment in which preneoplastic cells evolve to reach their full tumorigenic potential. Finally, we conclude that an in-depth understanding of DNA DSB repair pathways in human cells will lead to novel therapeutic strategies to treat cancer and potentially other human diseases.