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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.

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.

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.

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.

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.

The inability to protect stalled replication forks from nucleolytic degradation drives genome instability and is associated with 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 ssDNA gaps on the lagging strand interfere with the ASF1-CAF-1 pathway of nucleosome assembly, 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 its sequestration at inactive replication factories during replication stress. This CAF-1 recycling defect is caused by the accumulation of Polα-dependent 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 fork stability in a CAF-1-dependent manner. We also show that the activation of a HIRA-dependent compensatory histone deposition pathway restores fork stability to BRCA-deficient cells upon CAF-1 loss. We thus define nucleosome assembly as a critical determinant of BRCA-mediated fork stability. We further reveal lagging strand ssDNA gaps as drivers of fork degradation in BRCA-deficient cells, which operate by inhibiting PCNA unloading and CAF-1-dependent nucleosome assembly.

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. PCNA-K164 ubiquitination is critical for Okazaki fragment maturation and the activation of DNA damage tolerance pathways. Moreover, ubiquitinated PCNA operates in a fork protection pathway parallel to BRCA-RAD51. Whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. Utilizing PCNAK164R cells generated by CRISPR-Cas9, we demonstrate that this mutation causes DNA replication defects that impact origin activation. PCNAK164R cells accumulate single-stranded DNA gaps during replication that persist throughout mitosis due to compromised mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 to initiate MiDAS. Persistent gaps hence interfere with MCM2-7 double hexamer loading in the subsequent G1 phase. Our findings demonstrate that the impact of PCNAK164-Ub is not limited to S/G2 phases but extends to mitosis and G1 phase. PCNA-K164 ubiquitination promotes DNA gap filling during S/G2 phases of the cell cycle. This study identifies a novel role for K164 ubiquitination in replication dynamics and mitotic DNA synthesis and thus provides new insight into the players involved in counteracting under-replication.

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. This signal is crucial for Okazaki fragment (OF) maturation and for the activation of two DNA damage tolerance pathways; error-prone translesion synthesis and error-free template switching. Recently, we demonstrated that PCNA ubiquitination operates in a fork protection pathway parallel to BRCA-RAD51. However, whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. Utilizing PCNAK164R cells generated by CRISPR-Cas9 genome editing, we demonstrate that this mutation impacts origin licensing and causes DNA replication defects. Our data suggest that the accumulation of single-stranded (ss) DNA gaps from the previous replication cycle, interferes with the loading of MCM2-7 double hexamers in the following G1 phase. Insufficient origin licensing leads to under-replicated regions throughout the genome that are not resolved by mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 mono-ubiquitination to initiate MiDAS. Our findings demonstrate that the impact of PCNA-K164 ubiquitination is not limited to S/G2 phases but extends to G1 and mitosis. Competing Interest Statement The authors have declared no competing interest.

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 ubiquitination-deficient 293T and RPE1 cells generated through CRISPR/Cas9 genome editing, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-mediated 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 synthesis and maturation, thus interfering with efficient PCNA unloading by ATAD5 and subsequent nucleosomal deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and BRCA2 results in a synergistic increase in nascent DNA degradation and sensitivity to PARP-inhibitors. 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.

BRCA proteins are essential for Homologous Recombination DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response to chemotherapy of BRCA-deficient tumors is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which normally are subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 controls chemoresistance in BRCA2-deficient cells. We found that E2F7 depletion restores PARP inhibitor and cisplatin resistance in BRCA2-depleted cells. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7- mediated transcriptional repression, which enhances both Homologous Recombination DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of chemotherapy resistance in BRCA2-deficient cells, and identifies E2F7 as a novel biomarker for tumor response to PARP inhibitor therapy.