USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.

Conflicts with transcription make early replication late.

By sequencing sites of mitotic DNA synthesis in cells lacking homologous recombination, Groelly, Bhowmick, and colleagues show how conflicts between transcription and replication in early S phase can cause under-replicated DNA to persist into mitosis.

Sources, resolution and physiological relevance of R-loops and RNA-DNA hybrids.

RNA-DNA hybrids are generated during transcription, DNA replication and DNA repair and are crucial intermediates in these processes. When RNA-DNA hybrids are stably formed in double-stranded DNA, they displace one of the DNA strands and give rise to a three-stranded structure called an R-loop. R-loops are widespread in the genome and are enriched at active genes. R-loops have important roles in regulating gene expression and chromatin structure, but they also pose a threat to genomic stability, especially during DNA replication. To keep the genome stable, cells have evolved a slew of mechanisms to prevent aberrant R-loop accumulation. Although R-loops can cause DNA damage, they are also induced by DNA damage and act as key intermediates in DNA repair such as in transcription-coupled repair and RNA-templated DNA break repair. When the regulation of R-loops goes awry, pathological R-loops accumulate, which contributes to diseases such as neurodegeneration and cancer. In this Review, we discuss the current understanding of the sources of R-loops and RNA-DNA hybrids, mechanisms that suppress and resolve these structures, the impact of these structures on DNA repair and genome stability, and opportunities to therapeutically target pathological R-loops.

Hypertranscription and replication stress in cancer.

Replication stress results from obstacles to replication fork progression, including ongoing transcription, which can cause transcription-replication conflicts. Oncogenic signaling can promote global increases in transcription activity, also termed hypertranscription. Despite the widely accepted importance of oncogene-induced hypertranscription, its study remains neglected compared with other causes of replication stress and genomic instability in cancer. A growing number of recent studies are reporting that oncogenes, such as RAS, and targeted cancer treatments, such as bromodomain and extraterminal motif (BET) bromodomain inhibitors, increase global transcription, leading to R-loop accumulation, transcription-replication conflicts, and the activation of replication stress responses. Here we discuss our mechanistic understanding of hypertranscription-induced replication stress and the resulting cellular responses, in the context of oncogenes and targeted cancer therapies.

MYBL2 and ATM suppress replication stress in pluripotent stem cells.

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR-dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand-fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2-MRN-ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin.

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts.

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

Adenovirus E1B 55-Kilodalton Protein Targets SMARCAL1 for Degradation during Infection and Modulates Cellular DNA Replication.

Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.

BET Inhibition Induces HEXIM1- and RAD51-Dependent Conflicts between Transcription and Replication.

BET bromodomain proteins are required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on processes such as DNA replication will be important for future clinical applications. Here, we show that BET inhibition, and specifically inhibition of BRD4, causes replication stress through a rapid overall increase in RNA synthesis. We provide evidence that BET inhibition acts by releasing P-TEFb from its inhibitor HEXIM1, promoting interference between transcription and replication. Unusually, these transcription-replication conflicts do not activate the ATM/ATR-dependent DNA damage response but recruit the homologous recombination factor RAD51. Both HEXIM1 and RAD51 promote BET inhibitor-induced fork slowing but also prevent a DNA damage response. Our data suggest that BET inhibitors slow replication through concerted action of transcription and recombination machineries and shed light on the importance of replication stress in the action of this class of experimental cancer drugs.

Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells.

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.

MYBL2 Supports DNA Double Strand Break Repair in Hematopoietic Stem Cells.

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by blood cytopenias that occur as a result of somatic mutations in hematopoietic stem cells (HSC). MDS leads to ineffective hematopoiesis, and as many as 30% of patients progress to acute myeloid leukemia (AML). The mechanisms by which mutations accumulate in HSC during aging remain poorly understood. Here we identify a novel role for MYBL2 in DNA double-strand break (DSB) repair in HSC. In patients with MDS, low MYBL2 levels associated with and preceded transcriptional deregulation of DNA repair genes. Stem/progenitor cells from these patients display dysfunctional DSB repair kinetics after exposure to ionizing radiation (IR). Haploinsufficiency of Mybl2 in mice also led to a defect in the repair of DSBs induced by IR in HSC and was characterized by unsustained phosphorylation of the ATM substrate KAP1 and telomere fragility. Our study identifies MYBL2 as a crucial regulator of DSB repair and identifies MYBL2 expression levels as a potential biomarker to predict cellular response to genotoxic treatments in MDS and to identify patients with defects in DNA repair. Such patients with worse prognosis may require a different therapeutic regimen to prevent progression to AML.Significance: These findings suggest MYBL2 levels may be used as a biological biomarker to determine the DNA repair capacity of hematopoietic stem cells from patients with MDS and as a clinical biomarker to inform decisions regarding patient selection for treatments that target DNA repair.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5767/F1.large.jpg Cancer Res; 78(20); 5767-79. ©2018 AACR.

Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place.

Oncogene activation disturbs cellular processes and accommodates a complex landscape of changes in the genome that contribute to genomic instability, which accelerates mutation rates and promotes tumorigenesis. Part of this cellular turmoil involves deregulation of physiologic DNA replication, widely described as replication stress. Oncogene-induced replication stress is an early driver of genomic instability and is attributed to a plethora of factors, most notably aberrant origin firing, replication-transcription collisions, reactive oxygen species, and defective nucleotide metabolism.Significance: Replication stress is a fundamental step and an early driver of tumorigenesis and has been associated with many activated oncogenes. Deciphering the mechanisms that contribute to the replication stress response may provide new avenues for targeted cancer treatment. In this review, we discuss the latest findings on the DNA replication stress response and examine the various mechanisms through which activated oncogenes induce replication stress. Cancer Discov; 8(5); 537-55. ©2018 AACR.

PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation.

PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and by stabilising replication forks that encounter BER intermediates.

Increased global transcription activity as a mechanism of replication stress in cancer.

Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells.

TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATM-defective CLL that warrants clinical investigation.

Synthetic lethality in chronic lymphocytic leukaemia with DNA damage response defects by targeting the ATR pathway.

BACKGROUND: DNA damage response (DDR) defects, particularly TP53 and biallelic ataxia telangiectasia mutated (ATM) aberrations, are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukaemia (CLL). Therapies capable of providing long-term disease control in CLL patients with DDR defects are lacking. Using AZD6738, a novel ATR inhibitor, we investigated ATR pathway inhibition as a synthetically lethal strategy for targeting CLL cells with these defects. METHODS: The effect of AZD6738 was assessed by western blotting and immunofluorescence of key DDR proteins. Cytotoxicity was assessed by CellTiter-Gloluminescence assay (Promega, Madison, WI, USA) and by propidium iodide exclusion. Primary CLL cells with biallelic TP53 or ATM inactivation were xenotransplanted into NOD/Shi-scid/IL-2Rγ mice. After treatment with AZD6738 or vehicle, tumour load was measured by flow cytometric analysis of infiltrated spleens, and subclonal composition by fluorescence in-situ hybridisation for 17p(TP53) or 11q(ATM) deletion. FINDINGS: AZD6738 provided potent and specific inhibition of ATR signalling with compensatory activation of ATM/p53 pathway in cycling CLL cells in the presence of genotoxic stress. In p53 or ATM defective cells, AZD6738 treatment resulted in replication fork stalls and accumulation of unrepaired DNA damage, as evidenced by γH2AX and 53BP1 foci formation, which was carried through into mitosis, resulting in cell death by mitotic catastrophe. AZD6738 displayed selective cytotoxicity towards ATM or p53 deficient CLL cells, and was highly synergistic in combination with cytotoxic chemotherapy. This finding was confirmed in primary xenograft models of DDR-defective CLL, where treatment with AZD6738 resulted in decreased tumour load and selective reduction of CLL subclones with ATM or TP53 alterations. INTERPRETATION: We have provided mechanistic insight and demonstrated in-vitro and in-vivo efficacy of a novel therapeutic approach that specifically targets p53-null or ATM-null CLL cells. Such an approach can potentially help to avert clonal evolution, a major cause of therapeutic resistance and disease relapse. FUNDING: Leukaemia & Lymphoma Research.

Cancer therapy and replication stress: forks on the road to perdition.

Deregulated DNA replication occurs in cancer where it contributes to genomic instability. This process is a target of cytotoxic therapies. Chemotherapies exploit high DNA replication in cancer cells by modifying the DNA template or by inhibiting vital enzymatic activities that lead to slowing or stalling replication fork progression. Stalled replication forks can be converted into toxic DNA double-strand breaks resulting in cell death, i.e., replication stress. While likely crucial for many cancer treatments, replication stress is poorly understood due to its complexity. While we still know relatively little about the role of replication stress in cancer therapy, technical advances in recent years have shed new light on the effect that cancer therapeutics have on replication forks and the molecular mechanisms that lead from obstructed fork progression to cell death. This chapter will give an overview of our current understanding of replication stress in the context of cancer therapy.

Human PIF1 helicase supports DNA replication and cell growth under oncogenic-stress.

Unwinding duplex DNA is a critical processing step during replication, repair and transcription. Pif1 are highly conserved non-processive 5'->3' DNA helicases with well-established roles in maintenance of yeast genome stability. However, the function of the sole member of Pif1 family in humans remains unclear. Human PIF1 is essential for tumour cell viability, particularly during replication stress, but is dispensable in non-cancerous cells and Pif1 deficient mice. Here we report that suppression of PIF1 function slows replication fork rates and increases arrested forks during normal cycling conditions. Importantly, PIF1-dependent replication impediments impair S-phase progression and reduce proliferation rates of RAS oncogene-transformed fibroblasts, where replication fork slowing is exacerbated, but not parental, non-cancerous cells. Disrupted fork movement upon PIF1-depletion does not enhance double-stranded break formation or DNA damage responses but affects resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and mainly S-phase entry. Taken together, we characterised a functional role for human PIF1 in DNA replication that becomes important for cell growth under oncogenic stress. Given that oncogenes induce high levels of replication stress during the early stages of tumorigenesis, this function of PIF1 could become critical during cancer development.

BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors.