USP1-dependent nucleolytic expansion of PRIMPOL-generated nascent DNA strand discontinuities during replication stress.

DNA replication stress-induced fork arrest represents a significant threat to genomic integrity. One major mechanism of replication restart involves repriming downstream of the arrested fork by PRIMPOL, leaving behind a single-stranded DNA (ssDNA) gap. Accumulation of nascent strand ssDNA gaps has emerged as a possible determinant of the cellular hypersensitivity to genotoxic agents in certain genetic backgrounds such as BRCA deficiency, but how gaps are converted into cytotoxic structures is still unclear. Here, we investigate the processing of PRIMPOL-dependent ssDNA gaps upon replication stress induced by hydroxyurea and cisplatin. We show that gaps generated in PRIMPOL-overexpressing cells are expanded in the 3'-5' direction by the MRE11 exonuclease, and in the 5'-3' direction by the EXO1 exonuclease. This bidirectional exonucleolytic gap expansion ultimately promotes their conversion into DSBs. We moreover identify the de-ubiquitinating enzyme USP1 as a critical regulator of PRIMPOL-generated ssDNA gaps. USP1 promotes gap accumulation during S-phase, and their expansion by the MRE11 and EXO1 nucleases. This activity of USP1 is linked to its role in de-ubiquitinating PCNA, suggesting that PCNA ubiquitination prevents gap accumulation during replication. Finally, we show that USP1 depletion suppresses DSB formation in PRIMPOL-overexpressing cells, highlighting an unexpected role for USP1 in promoting genomic instability under these conditions.

Loss of MED12 activates the TGFß pathway to promote chemoresistance and replication fork stability in BRCA-deficient cells.

Understanding chemoresistance mechanisms in BRCA-deficient cells will allow for identification of biomarkers for predicting tumor response to therapy, as well as the design of novel therapeutic approaches targeting this chemoresistance. Here, we show that the protein MED12, a component of the Mediator transcription regulation complex, plays an unexpected role in regulating chemosensitivity in BRCA-deficient cells. We found that loss of MED12 confers resistance to cisplatin and PARP inhibitors in both BRCA1- and BRCA2-deficient cells, which is associated with restoration of both homologous recombination and replication fork stability. Surprisingly, MED12-controlled chemosensitivity does not involve a function of the Mediator complex, but instead reflects a distinct role of MED12 in suppression of the TGFß pathway. Importantly, we show that ectopic activation of the TGFß pathway is enough to overcome the fork protection and DNA repair defects of BRCA-mutant cells, resulting in chemoresistance. Our work identifies the MED12-TGFß module as an important regulator of genomic stability and chemosensitivity in BRCA-deficient cells.

Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.

The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFß signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.

Heterozygous RNF13 Gain-of-Function Variants Are Associated with Congenital Microcephaly, Epileptic Encephalopathy, Blindness, and Failure to Thrive.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates a stress response mechanism to clear out the unfolded proteins by either facilitating their re-folding or inducing their degradation. When this fails, an apoptotic cascade is initiated so that the affected cell is eliminated. IRE1α is a critical sensor of the unfolded-protein response, essential for initiating the apoptotic signaling. Here, we report an infantile neurodegenerative disorder associated with enhanced activation of IRE1α and increased apoptosis. Three unrelated affected individuals with congenital microcephaly, infantile epileptic encephalopathy, and profound developmental delay were found to carry heterozygous variants (c.932T>C [p.Leu311Ser] or c.935T>C [p.Leu312Pro]) in RNF13, which codes for an IRE1α-interacting protein. Structural modeling predicted that the variants, located on the surface of the protein, would not alter overall protein folding. Accordingly, the abundance of RNF13 and IRE1α was not altered in affected individuals' cells. However, both IRE1α-mediated stress signaling and stress-induced apoptosis were increased in affected individuals' cells. These results indicate that the RNF13 variants confer gain of function to the encoded protein and thereby lead to altered signaling of the ER stress response associated with severe neurodegeneration in infancy.

Genome-wide CRISPR synthetic lethality screen identifies a role for the ADP-ribosyltransferase PARP14 in DNA replication dynamics controlled by ATR.

The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells.

DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.

Heterozygous De Novo UBTF Gain-of-Function Variant Is Associated with Neurodegeneration in Childhood.

Ribosomal RNA (rRNA) is transcribed from rDNA by RNA polymerase I (Pol I) to produce the 45S precursor of the 28S, 5.8S, and 18S rRNA components of the ribosome. Two transcription factors have been defined for Pol I in mammals, the selectivity factor SL1, and the upstream binding transcription factor (UBF), which interacts with the upstream control element to facilitate the assembly of the transcription initiation complex including SL1 and Pol I. In seven unrelated affected individuals, all suffering from developmental regression starting at 2.5-7 years, we identified a heterozygous variant, c.628G>A in UBTF, encoding p.Glu210Lys in UBF, which occurred de novo in all cases. While the levels of UBF, Ser388 phosphorylated UBF, and other Pol I-related components (POLR1E, TAF1A, and TAF1C) remained unchanged in cells of an affected individual, the variant conferred gain of function to UBF, manifesting by markedly increased UBF binding to the rDNA promoter and to the 5'- external transcribed spacer. This was associated with significantly increased 18S expression, and enlarged nucleoli which were reduced in number per cell. The data link neurodegeneration in childhood with altered rDNA chromatin status and rRNA metabolism.

PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress.

During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation, and its overexpression alleviates cellular sensitivity to replication stress and fosters the restart of stalled replication forks. Importantly, mouse xenograft studies showed that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation, potentially by alleviating replication stress and suggest that targeting PARP10 may represent a novel therapeutic approach.

Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells.

BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and 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 can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.

HUWE1 interacts with PCNA to alleviate replication stress.

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S-phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT-type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA We show that HUWE1-knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono-ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.

A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress.

Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.

PARP10 deficiency manifests by severe developmental delay and DNA repair defect.

DNA repair mechanisms such as nucleotide excision repair (NER) and translesion synthesis (TLS) are dependent on proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory protein. Recently, homozygosity for p.Ser228Ile mutation in the PCNA gene was reported in patients with neurodegeneration and impaired NER. Using exome sequencing, we identified a homozygous deleterious mutation, c.648delAG, in the PARP10 gene, in a patient suffering from severe developmental delay. In agreement, PARP10 protein was absent from the patient cells. We have previously shown that PARP10 is recruited by PCNA to DNA damage sites and is required for DNA damage resistance. The patient cells were significantly more sensitive to hydroxyurea and UV-induced DNA damage than control cells, resulting in increased apoptosis, indicating DNA repair impairment in the patient cells. PARP10 deficiency joins the long list of DNA repair defects associated with neurodegenerative disorders, including ataxia telangiectasia, xeroderma pigmentosum, Cockayne syndrome, and the recently reported PCNA mutation.

The ADP-ribosyltransferase PARP10/ARTD10 interacts with proliferating cell nuclear antigen (PCNA) and is required for DNA damage tolerance.

All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.

Role of Translesion DNA Synthesis in the Metabolism of Replication-associated Nascent Strand Gaps.

Translesion DNA synthesis (TLS) is a DNA damage tolerance pathway utilized by cells to overcome lesions encountered throughout DNA replication. During replication stress, cancer cells show increased dependency on TLS proteins for cellular survival and chemoresistance. TLS proteins have been described to be involved in various DNA repair pathways. One of the major emerging roles of TLS is single-stranded DNA (ssDNA) gap-filling, primarily after the repriming activity of PrimPol upon encountering a lesion. Conversely, suppression of ssDNA gap accumulation by TLS is considered to represent a mechanism for cancer cells to evade the toxicity of chemotherapeutic agents, specifically in BRCA-deficient cells. Thus, TLS inhibition is emerging as a potential treatment regimen for DNA repair-deficient tumors.

PARP10 promotes the repair of nascent strand DNA gaps through RAD18 mediated translesion synthesis.

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.

CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single stranded DNA gaps.

Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is however imperfect, and continuously evolving. Recent advances in the field highlighted single stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon the arrest of replication forks at sites of cisplatin adducts, and restart of DNA synthesis downstream of the lesion through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wildtype cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway, and show that RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing, shedding light on the mechanisms of cisplatin sensitization in cancer therapy.

Mono-ADP-ribosylation by PARP10 and PARP14 in genome stability.

ADP-ribosylation is a post-translational modification involved in a variety of processes including DNA damage repair, transcriptional regulation, and cellular proliferation. Depending on the number of ADP moieties transferred to target proteins, ADP-ribosylation can be classified either as mono-ADP-ribosylation (MARylation) or poly-ADP-ribosylation (PARylation). This post-translational modification is catalyzed by enzymes known as ADP-ribosyltransferases (ARTs), which include the poly (ADP-ribose)-polymerase (PARP) superfamily of proteins. Certain members of the PARP family including PARP1 and PARP2 have been extensively studied and assessed as therapeutic targets. However, the other members of the PARP family of protein are not as well studied but have gained attention in recent years given findings suggesting their roles in an increasing number of cellular processes. Among these other members are PARP10 and PARP14, which have gradually emerged as key players in maintenance of genomic stability and carcinogenesis. PARP10 and PARP14 catalyze the transfer of a single ADP moiety to target proteins. Here, we summarize the current knowledge on MARylation in DNA repair and cancer, focusing on PARP10 and PARP14. We highlight the roles of PARP10 and PARP14 in cancer progression and response to chemotherapeutics and briefly discuss currently known PARP10 and PARP14 inhibitors.

Multi-step processing of replication stress-derived nascent strand DNA gaps by MRE11 and EXO1 nucleases.

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.