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Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors. PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.

Suppression of nascent DNA degradation has emerged as an essential role of the BRCA pathway in genome protection. In BRCA-deficient cells, the MRE11 nuclease is responsible for both resection of reversed replication forks, and accumulation of single stranded DNA gaps behind forks. Here, we show that the mono-ADP-ribosyltransferase PARP14 is a critical co-factor of MRE11. PARP14 is recruited to nascent DNA upon replication stress in BRCA-deficient cells, and through its catalytic activity, mediates the engagement of MRE11. Loss or inhibition of PARP14 suppresses MRE11-mediated fork degradation and gap accumulation, and promotes genome stability and chemoresistance of BRCA-deficient cells. Moreover, we show that the KU complex binds reversed forks and protects them against EXO1-catalyzed degradation. KU recruits the PARP14-MRE11 complex, which initiates partial resection to release KU and allow long-range resection by EXO1. Our work identifies a multistep process of nascent DNA processing at stalled replication forks in BRCA-deficient cells. Protection of replication forks against nucleolytic degradation is crucial for genome stability. Here, Dhoonmoon et al identify PARP14 and the KU complex as essential regulators of fork degradation by MRE11 and EXO1 nucleases in BRCA-deficient cells.

Accumulation of single stranded DNA (ssDNA) gaps in the nascent strand during DNA replication has been associated with cytotoxicity and hypersensitivity to genotoxic stress, particularly upon inactivation of the BRCA tumor suppressor pathway. However, how ssDNA gaps contribute to genotoxicity is not well understood. Here, we describe a multi-step nucleolytic processing of replication stress-induced ssDNA gaps which converts them into cytotoxic double stranded DNA breaks (DSBs). We show that ssDNA gaps are extended bidirectionally by MRE11 in the 3’−5’ direction and by EXO1 in the 5’−3’ direction, in a process which is suppressed by the BRCA pathway. Subsequently, the parental strand at the ssDNA gap is cleaved by the MRE11 endonuclease generating a double strand break. We also show that exposure to bisphenol A (BPA) and diethylhexyl phthalate (DEHP), which are widespread environmental contaminants due to their use in plastics manufacturing, causes nascent strand ssDNA gaps during replication. These gaps are processed through the same mechanism described above to generate DSBs. Our work sheds light on both the relevance of ssDNA gaps as major determinants of genomic instability, as well as the mechanism through which they are processed to generate genomic instability and cytotoxicity. This study describes a multi-step nucleolytic processing of ssDNA gaps which converts them into cytotoxic double stranded breaks. Gaps can be induced not only by replication stress or genotoxic chemotherapy, but also by exposure to the environmental contaminant bisphenol A.

Cyclin D1 is an essential regulator of the G1–S cell-cycle transition and is overexpressed in many cancers. Expression of cyclin D1 is under tight cellular regulation that is controlled by many signaling pathways. Here we report that PARP14, a member of the poly(ADP-ribose) polymerase (PARP) family, is a regulator of cyclin D1 expression. Depletion of PARP14 leads to decreased cyclin D1 protein levels. In cells with a functional retinoblastoma (RB) protein pathway, this results in G1 cell-cycle arrest and reduced proliferation. Mechanistically, we found that PARP14 controls cyclin D1 mRNA levels. Using luciferase assays, we show that PARP14 specifically regulates cyclin D1 3′UTR mRNA stability. Finally, we also provide evidence that G1 arrest in PARP14-depleted cells is dependent on an intact p53–p21 pathway. Our work uncovers a new role for PARP14 in promoting cell-cycle progression through both cyclin D1 and the p53 pathway.

DNA repair genes are generally considered tumor suppressors, as their inactivation is observed in tumors and is associated with carcinogenesis. Mutations in BRCA1 and BRCA2 genes are observed in breast, ovarian, and other cancers. This results in defective homologous recombination DNA repair, as well as in degradation of nascent DNA during replication stress, catalyzed by exonucleases including EXO1 and MRE11. However, most tumors are BRCA pathway-proficient. Here, we show that EXO1 is overexpressed in a significant proportion of tumors. EXO1 overexpression causes the degradation of nascent DNA at both single stranded DNA (ssDNA) gaps and reversed replication forks. Importantly, this degradation occurs efficiently in BRCA-proficient cells, through cooperation with MRE11. This results in increased double strand break formation and hypersensitivity to genotoxic agents. We thus identify increased EXO1 activity as a mechanism of genomic instability similar to BRCA pathway inactivation, but occurring more frequently in tumors compared to BRCA inactivation. DNA repair genes are generally considered tumor suppressors, as they maintain genomic stability. Here, the authors show that the exonuclease EXO1 is overexpressed in a significant proportion of tumors. EXO1 overexpression results in genomic instability through the degradation of nascent DNA.

Replication stress compromises genomic integrity. Fork blocking lesions such as those induced by cisplatin and other chemotherapeutic agents arrest replication forks. Repriming downstream of these lesions represents an important mechanism of replication restart, however the single stranded DNA (ssDNA) gaps left behind, unless efficiently filled, can serve as entry point for nucleases. Nascent strand gaps can be repaired by BRCA-mediated homology repair. Alternatively, gaps can also be filled by translesion synthesis (TLS) polymerases. How these events are regulated is still not clear. Here, we show that PARP10, a poorly-characterized mono-ADP-ribosyltransferase, is recruited to nascent strand gaps to promote their repair. PARP10 interacts with the ubiquitin ligase RAD18 and recruits it to these structures, resulting in the ubiquitination of the replication factor PCNA. PCNA ubiquitination, in turn, recruits the TLS polymerase REV1 for gap filling. We show that PARP10 recruitment to gaps and the subsequent REV1-mediated gap filling requires both the catalytic activity of PARP10, and its ability to interact with PCNA. We moreover show that PARP10 is hyperactive in BRCA-deficient cells, and its inactivation potentiates gap accumulations and cytotoxicity in these cells. Our work uncovers PARP10 as a regulator of ssDNA gap filling, which promotes genomic stability in BRCA-deficient cells. ssDNA gaps have emerged as structures which may mediate cytotoxicity of chemotherapeutic agents such as cisplatin. Here, the authors show that the mono-ADPribosyltransferase PARP10 promotes the repair of ssDNA gaps by recruiting RAD18 to initiate PCNA ubiquitination-mediated translesion synthesis.

The tumor suppressor BRCA2 protects stalled forks from degradation to maintain genome stability. However, the molecular mechanism(s) whereby unprotected forks are stabilized remains to be fully characterized. Here, we demonstrate that WRN helicase ensures efficient restart and limits excessive degradation of stalled forks in BRCA2-deficient cancer cells. In vitro, WRN ATPase/helicase catalyzes fork restoration and curtails MRE11 nuclease activity on regressed forks. We show that WRN helicase inhibitor traps WRN on chromatin leading to rapid fork stalling and nucleolytic degradation of unprotected forks by MRE11, resulting in MUS81-dependent double-strand breaks, elevated non-homologous end-joining and chromosomal instability. WRN helicase inhibition reduces viability of BRCA2-deficient cells and potentiates cytotoxicity of a poly (ADP)ribose polymerase (PARP) inhibitor. Furthermore, BRCA2-deficient xenograft tumors in mice exhibited increased DNA damage and growth inhibition when treated with WRN helicase inhibitor. This work provides mechanistic insight into stalled fork stabilization by WRN helicase when BRCA2 is deficient. The tumor suppressor BRCA2 protects stalled DNA replication forks from unrestrained degradation; however the mechanism whereby unprotected stalled forks are preserved and restarted has remained elusive. Here the authors show that the WRN helicase promotes stalled fork recovery and limits fork hyper-degradation in the absence of BRCA2 protection.

The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and underlies chemosensitivity in BRCA-deficient tumors. An emerging hallmark of BRCA-deficiency is the inability to suppress replication-associated single-stranded DNA (ssDNA) gaps. Here, we report that lagging strand ssDNA gaps interfere with the ASF1-CAF-1 nucleosome assembly pathway, and drive fork degradation in BRCA-deficient cells. We show that CAF-1 function at replication forks is lost in BRCA-deficient cells, due to defects in its recycling during replication stress. This CAF-1 recycling defect is caused by lagging strand gaps which preclude PCNA unloading, causing sequestration of PCNA-CAF-1 complexes on chromatin. Importantly, correcting PCNA unloading defects in BRCA-deficient cells restores CAF-1-dependent fork stability. We further show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells. We thus define lagging strand gap suppression and nucleosome assembly as critical enablers of BRCA-mediated fork stability. Efficient DNA replication is crucial for genome stability. Here, Thakar et al. report that accumulation of lagging strand ssDNA gaps during replication interferes with nucleosome assembly and drives replication fork degradation in BRCA-deficient cells.

Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells. Mutations in the homologous recombination proteins BRCA1 and BRCA2 can sensitize cells to treatment with inhibitors of poly-ADP-ribose polymerase 1 (PARPi), but resistance to the treatment can occur. Here the authors by genome-wide CRISPR knockout and activation screens reveal novel pathways of PARPi resistance in BRCA2-deficient cells.

Transient receptor potential channel melastatin 2 (TRPM2) is highly expressed in cancer and has an essential function in preserving viability through maintenance of mitochondrial function and antioxidant response. Here, the role of TRPM2 in cell survival was examined in neuroblastoma cells with TRPM2 deletion with CRISPR technology. Viability was significantly decreased in TRPM2 knockout after doxorubicin treatment. RNA sequence analysis and RT-qPCR revealed reduced RNAs encoding master transcription regulators FOXM1 and E2F1/2 and downstream cell cycle targets including Cyclin B1, CDK1, PLK1, and CKS1. CHIP analysis demonstrated decreased FOXM1 binding to their promoters. Western blotting confirmed decreased expression, and increased expression of CDK inhibitor p21, a CKS1 target. In cells with TRPM2 deletion, cell cycle progression to S and G2/M phases was reduced after treatment with doxorubicin. RNA sequencing also identified decreased DNA repair proteins in cells with TRPM2 deletion after doxorubicin treatment, and DNA damage was increased. Wild type TRPM2, but not Ca 2+ -impermeable mutant E960D, restored live cell number and reconstituted expression of E2F1, FOXM1, and cell cycle/DNA repair proteins. FOXM1 expression alone restored viability. TRPM2 is a potential therapeutic target to reduce tumor proliferation and increase doxorubicin sensitivity through modulation of FOXM1, E2F1, and cell cycle/DNA repair proteins.