The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.

TGF-β secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-β-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-β-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution.

The Set8/PR-Set7/KMT5a methyltransferase plays critical roles in governing transcriptional regulation, cell cycle progression and tumorigenesis. Although CRL4(Cdt2) was reported to regulate Set8 stability, deleting the PIP motif only led to partial resistance to ultraviolet-induced degradation of Set8, indicating the existence of additional E3 ligase(s) controlling Set8 stability. Furthermore, it remains largely undefined how DNA damage-induced kinase cascades trigger the timely destruction of Set8 to govern tumorigenesis. Here, we report that SCF(β-TRCP) earmarks Set8 for ubiquitination and degradation in a casein kinase I-dependent manner, which is activated by DNA-damaging agents. Biologically, both CRL4(Cdt2) and SCF(β-TRCP)-mediated pathways contribute to ultraviolet-induced Set8 degradation to control cell cycle progression, governing the onset of DNA damage-induced checkpoints. Therefore, like many critical cell cycle regulators including p21 and Cdt1, we uncover a tight regulatory network to accurately control Set8 abundance. Our studies further suggest that aberrancies in this delicate degradation pathway might contribute to aberrant elevation of Set8 in human tumours.

Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.

Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.

Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarcoma, as well as its potential to be a therapeutic target.

Mutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

Current targeted cancer therapies are largely guided by mutations of a single gene, which overlooks concurrent genomic alterations. Here, we show that RNASEH2B, RB1, and BRCA2, three closely located genes on chromosome 13q, are frequently deleted in prostate cancer individually or jointly. Loss of RNASEH2B confers cancer cells sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition due to impaired ribonucleotide excision repair and PARP trapping. When co-deleted with RB1, however, cells lose their sensitivity, in part, through E2F1-induced BRCA2 expression, thereby enhancing homologous recombination repair capacity. Nevertheless, loss of BRCA2 resensitizes RNASEH2B/RB1 co-deleted cells to PARP inhibition. Our results may explain some of the disparate clinical results from PARP inhibition due to interaction between multiple genomic alterations and support a comprehensive genomic test to determine who may benefit from PARP inhibition. Last, we show that ATR inhibition can disrupt E2F1-induced BRCA2 expression and overcome PARP inhibitor resistance caused by RB1 loss.

Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C terminus (BRCT) domains interact with multiple proteins and are required for BRCA1's tumor-suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients.

Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe an assay to visualize ALT-mediated telomeric DNA synthesis in ALT-associated PML bodies (APBs) without DNA-damaging agents or replication inhibitors. Using this assay, we find that ALT occurs through two distinct mechanisms. One of the ALT mechanisms requires RAD52, a protein implicated in break-induced DNA replication (BIR). We demonstrate that RAD52 directly promotes telomeric D-loop formation in vitro and is required for maintaining telomeres in ALT-positive cells. Unexpectedly, however, RAD52 is dispensable for C-circle formation, a hallmark of ALT. In RAD52-knockout ALT cells, C-circle formation and RAD52-independent ALT DNA synthesis gradually increase as telomeres are shortened, and these activities are dependent on BLM and BIR proteins POLD3 and POLD4. These results suggest that ALT occurs through a RAD52-dependent and a RAD52-independent BIR pathway, revealing the bifurcated framework and dynamic nature of this process.

APOBEC3A is a cytidine deaminase driving mutagenesis, DNA replication stress and DNA damage in cancer cells. While the APOBEC3A-induced vulnerability of cancers offers an opportunity for therapy, APOBEC3A protein and mRNA are difficult to quantify in tumors due to their low abundance. Here, we describe a quantitative and sensitive assay to measure the ongoing activity of APOBEC3A in tumors. Using hotspot RNA mutations identified from APOBEC3A-positive tumors and droplet digital PCR, we develop an assay to quantify the RNA-editing activity of APOBEC3A. This assay is superior to APOBEC3A protein- and mRNA-based assays in predicting the activity of APOBEC3A on DNA. Importantly, we demonstrate that the RNA mutation-based APOBEC3A assay is applicable to clinical samples from cancer patients. Our study presents a strategy to follow the dysregulation of APOBEC3A in tumors, providing opportunities to investigate the role of APOBEC3A in tumor evolution and to target the APOBEC3A-induced vulnerability in therapy.

Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.

The cyclic GMP-AMP synthase (cGAS), a sensor of cytosolic DNA, is critical for the innate immune response. Here, we show that loss of cGAS in untransformed and cancer cells results in uncontrolled DNA replication, hyperproliferation, and genomic instability. While the majority of cGAS is cytoplasmic, a fraction of cGAS associates with chromatin. cGAS interacts with replication fork proteins in a DNA binding-dependent manner, suggesting that cGAS encounters replication forks in DNA. Independent of cGAMP and STING, cGAS slows replication forks by binding to DNA in the nucleus. In the absence of cGAS, replication forks are accelerated, but fork stability is compromised. Consequently, cGAS-deficient cells are exposed to replication stress and become increasingly sensitive to radiation and chemotherapy. Thus, by acting as a decelerator of DNA replication forks, cGAS controls replication dynamics and suppresses replication-associated DNA damage, suggesting that cGAS is an attractive target for exploiting the genomic instability of cancer cells.

The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation.BRCA proteins have emerged as key stabilizing factors for the maintenance of replication forks following replication stress. Here the authors describe how reversed replication forks are degraded in the absence of BRCA2, and a MUS81 and POLD3-dependent mechanism of rescue following the withdrawal of genotoxic agent.

Mutation signatures associated with apolipoprotein B mRNA editing catalytic polypeptide-like 3A/B (APOBEC3A/B) cytidine deaminases are prevalent across cancers, implying their roles as mutagenic drivers during tumorigenesis and tumor evolution. APOBEC3A (A3A) expression induces DNA replication stress and increases the cellular dependency on the ataxia telangiectasia and Rad3-related (ATR) kinase for survival. Nonetheless, how A3A induces DNA replication stress remains unclear. We show that A3A induces replication stress without slowing replication forks. We find that A3A induces single-stranded DNA (ssDNA) gaps through PrimPol-mediated repriming. A3A-induced ssDNA gaps are repaired by multiple pathways involving ATR, RAD51, and translesion synthesis. Both ATR inhibition and trapping of poly(ADP-ribose) polymerase (PARP) on DNA by PARP inhibitor impair the repair of A3A-induced gaps, preferentially killing A3A-expressing cells. When used in combination, PARP and ATR inhibitors selectively kill A3A-expressing cells synergistically in a manner dependent on PrimPol-generated gaps. Thus, A3A-induced replication stress arises from PrimPol-generated ssDNA gaps, which confer a therapeutic vulnerability to gap-targeted DNA repair inhibitors.

Homologous recombination (HR) is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB). However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.

Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.