bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒12‒13
27 papers selected by
Sean Rudd
Karolinska Institutet

  1. Mol Cell. 2020 Dec 01. pii: S1097-2765(20)30785-1. [Epub ahead of print]
      Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon replication stress, DNA topoisomerase IIalpha (TOP2A) is recruited to stalled forks in a manner dependent on the SNF2-family DNA translocases HLTF, ZRANB3, and SMARCAL1. This is accompanied by an increase in TOP2A SUMOylation mediated by the SUMO E3 ligase ZATT and followed by recruitment of a SUMO-targeted DNA translocase, PICH. Disruption of the ZATT-TOP2A-PICH axis results in accumulation of partially reversed forks and enhanced genome instability. These results suggest that fork reversal occurs via a sequential two-step process. First, HLTF, ZRANB3, and SMARCAL1 initiate limited fork reversal, creating superhelical strain in the newly replicated sister chromatids. Second, TOP2A drives extensive fork reversal by resolving the resulting topological barriers and via its role in recruiting PICH to stalled forks.
    Keywords:  DNA topoisomerase; DNA translocase; SUMO E3 ligase; SUMOylation; genome instability; replication fork restart; replication fork reversal
  2. Sci Adv. 2020 Dec;pii: eabc8257. [Epub ahead of print]6(50):
      Chromosome instability (CIN) underpins cancer evolution and is associated with drug resistance and poor prognosis. Understanding the mechanistic basis of CIN is thus a priority. The structure-specific endonuclease Mus81-Eme1 is known to prevent CIN. Intriguingly, however, here we show that the aberrant processing of late replication intermediates by Mus81-Eme1 is a source of CIN. Upon depletion of checkpoint kinase 1 (Chk1), Mus81-Eme1 cleaves under-replicated DNA engaged in mitotic DNA synthesis, leading to chromosome segregation defects. Supplementing cells with nucleosides allows the completion of mitotic DNA synthesis, restraining Mus81-Eme1-dependent DNA damage in mitosis and the ensuing CIN. We found no correlation between CIN arising from nucleotide shortage in mitosis and cell death, which were selectively linked to DNA damage load in mitosis and S phase, respectively. Our findings imply the possibility of optimizing Chk1-directed therapies by inducing cell death while curtailing CIN, a common side effect of chemotherapy.
  3. DNA Repair (Amst). 2020 Nov 26. pii: S1568-7864(20)30287-1. [Epub ahead of print]97 103027
      8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a major product of DNA oxidation, is a pre-mutagenic lesion which is prone to mispair, if left unrepaired, with 2'-deoxyadenosine during DNA replication. While unrepaired or incompletely repaired 8-oxodG has classically been associated with genome instability and cancer, it has recently been reported to have a role in the epigenetic regulation of gene expression. Despite the growing collection of genome-wide 8-oxodG mapping studies that have been used to provide new insight on the functional nature of 8-oxodG within the genome, a comprehensive view that brings together the epigenetic and the mutagenic nature of the 8-oxodG is still lacking. To help address this gap, this review aims to provide (i) a description of the state-of-the-art knowledge on both the mutagenic and epigenetic roles of 8-oxodG; (ii) putative molecular models through which the 8-oxodG can cause genome instability; (iii) a possible molecular model on how 8-oxodG, acting as an epigenetic signal, could cause the translocations and deletions which are associated with cancer.
    Keywords:  8-oxodG; Base excision repair; Cancer; Double-strand break; Genome instability; Single-Strand break; TOP1-DPC
  4. EMBO Rep. 2020 Dec 02. e50410
      DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.
    Keywords:  DNA damage tolerance; Rad52; homologous recombination; template switching; translesion synthesis
  5. Front Cell Dev Biol. 2020 ;8 607060
      8-Oxoguanine DNA glycosylase (OGG1) is the major cellular enzyme required for the excision of 8-oxoguanine DNA base lesions in DNA through the base excision repair (BER) pathway, and therefore plays a major role in suppressing mutagenesis and in controlling genome stability. However, the mechanism of regulation of cellular OGG1 protein, particularly in response to oxidative stress, is unclear. We have purified the major E3 ubiquitin ligase responsible for OGG1 ubiquitylation from human cell extracts, and identify this as E3 ubiquitin-protein ligase NEDD4-like (NEDD4L). We demonstrate that recombinant NEDD4L stimulates ubiquitylation of OGG1 in vitro, particularly on lysine 341, and that NEDD4L and OGG1 interact in U2OS cells. Depletion of NEDD4L in U2OS cells has no impact on the stability and steady-state protein levels of OGG1, however, OGG1 stability is enhanced in response to oxidative stress induced by ionizing radiation. Furthermore, ubiquitylation of OGG1 by NEDD4L in vitro inhibits its DNA glycosylase/lyase activity. As a consequence of prolonged OGG1 stability and increased excision activity in the absence of NEDD4L, cells display increased DNA repair capacity but conversely that this decreases cell survival post-irradiation. This effect can be reproduced following OGG1 overexpression, suggesting that dysregulation of OGG1 increases the formation of lethal intermediate DNA lesions. Our study therefore highlights the importance of balancing OGG1 protein levels and BER capacity in maintaining genome stability.
    Keywords:  DNA damage; DNA repair; NEDD4L; OGG1; ubiquitin
  6. Brief Funct Genomics. 2020 Dec 07. pii: elaa022. [Epub ahead of print]
      Post-translational modifications of proteins are well-established participants in DNA damage response (DDR) pathways, which function in the maintenance of genome integrity. Emerging evidence is starting to reveal the involvement of modifications on RNA in the DDR. RNA modifications are known regulators of gene expression but how and if they participate in DNA repair and genome maintenance has been poorly understood. Here, we review several studies that have now established RNA modifications as key components of DNA damage responses. RNA modifying enzymes and the binding proteins that recognize these modifications localize to and participate in the repair of UV-induced and DNA double-strand break lesions. RNA modifications have a profound effect on DNA-RNA hybrids (R-loops) at DNA damage sites, a structure known to be involved in DNA repair and genome stability. Given the importance of the DDR in suppressing mutations and human diseases such as neurodegeneration, immunodeficiencies, cancer and aging, RNA modification pathways may be involved in human diseases not solely through their roles in gene expression but also by their ability to impact DNA repair and genome stability.
    Keywords:  DNA damage; DNA repair; R-loops; RNA modification; genome integrity; transcription
  7. RNA. 2020 Dec 09. pii: rna.078519.120. [Epub ahead of print]
      In order to survive to the exposure of DNA damaging agents, cells activate a complex response that coordinates the cellular metabolism, cell cycle progression and DNA repair. Among many other events, recent evidence has described global changes in mRNA splicing in cells treated with genotoxic agents. Here, we explore further this DNA damage-dependent alternative splicing. Indeed, we show that both the splicing factor SF3B2 and the repair protein CtIP contribute to the global pattern of splicing both in cells treated or not to DNA damaging agents. Additionally, we focus on a specific DNA damage- and CtIP-dependent alternative splicing event of the helicase PIF1 and explore its relevance for the survival of cells upon exposure to ionizing radiation. Indeed, we described how the nuclear, active form of PIF1 is substituted by a splicing variant, named vPIF1, in a fashion that requires both the presence of DNA damage and CtIP. Interestingly, timely expression of vPIF1 is required for optimal survival to exposure to DNA damaging agents, but early expression of this isoform delays early events of the DNA damage response. On the contrary, expression of the full length PIF1 facilitates those early events, but increases the sensitivity to DNA damaging agents if the expression is maintained long-term.
    Keywords:  Alternative Splicing; CtIP; DNA Damage Response; SF3B
  8. Int J Cancer. 2020 Dec 07.
      Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified 7 proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing 13 C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.
    Keywords:  cancer cell metabolism; cell death; de novo serine synthesis pathway; one-carbon metabolism; therapy resistance
  9. Curr Opin Genet Dev. 2020 Dec 03. pii: S0959-437X(20)30145-3. [Epub ahead of print]67 41-51
      Repetitive sequences throughout the genome are a major source of endogenous DNA damage, due to the propensity of many of them to form alternative non-B DNA structures that can interfere with replication, transcription, and DNA repair. These repetitive sequences are prone to breakage (fragility) and instability (changes in repeat number). Repeat fragility and expansions are linked to several diseases, including many cancers and neurodegenerative diseases, hence the importance of understanding the mechanisms that cause genome instability and contribute to these diseases. This review focuses on recent findings of mechanisms causing repeat fragility and instability, new associations between repeat expansions and genetic diseases, and potential therapeutic options to target repeat expansions.
  10. Nucleic Acids Res. 2020 Dec 09. pii: gkaa1120. [Epub ahead of print]
      Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.
  11. Int J Mol Sci. 2020 Dec 04. pii: E9248. [Epub ahead of print]21(23):
      Cisplatin is a chemotherapeutic drug used for the treatment of a number of cancers. The efficacy of cisplatin relies on its binding to DNA and the induction of cytotoxic DNA damage to kill cancer cells. Cisplatin-based therapy is best known for curing testicular cancer; however, treatment of other solid tumors with cisplatin has not been as successful. Pre-clinical and clinical studies have revealed nucleotide excision repair (NER) as a major resistance mechanism against cisplatin in tumor cells. NER is a versatile DNA repair system targeting a wide range of helix-distorting DNA damage. The NER pathway consists of multiple steps, including damage recognition, pre-incision complex assembly, dual incision, and repair synthesis. NER proteins can recognize cisplatin-induced DNA damage and remove the damage from the genome, thereby neutralizing the cytotoxicity of cisplatin and causing drug resistance. Here, we review the molecular mechanism by which NER repairs cisplatin damage, focusing on the recent development of genome-wide cisplatin damage mapping methods. We also discuss how the expression and somatic mutations of key NER genes affect the response of cancer cells to cisplatin. Finally, small molecules targeting NER factors provide important tools to manipulate NER capacity in cancer cells. The status of research on these inhibitors and their implications in cancer treatment will be discussed.
    Keywords:  CSB; DNA damage; ERCC1-XPF; XPA; chemotherapy
  12. DNA Repair (Amst). 2020 Nov 22. pii: S1568-7864(20)30278-0. [Epub ahead of print]97 103018
      Homologous recombination (HR), considered the highest fidelity DNA double-strand break (DSB) repair pathway that a cell possesses, is capable of repairing multiple DSBs without altering genetic information. However, in "last resort" scenarios, HR can be directed to low fidelity subpathways which often use non-allelic donor templates. Such repair mechanisms are often highly mutagenic and can also yield chromosomal rearrangements and/or deletions. While the choice between HR and its less precise counterpart, non-homologous end joining (NHEJ), has received much attention, less is known about how cells manage and prioritize HR subpathways. In this review, we describe work focused on how chromatin and nuclear architecture orchestrate subpathway choice and repair template usage to maintain genome integrity without sacrificing cell survival. Understanding the relationships between nuclear architecture and recombination mechanics will be critical to understand these cellular repair decisions.
    Keywords:  Chromatin; DNA repair; Genome integrity; Homologous recombination; Nuclear architecture; Pathway choice
  13. Nucleic Acids Res. 2020 Dec 08. pii: gkaa1153. [Epub ahead of print]
      8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3'-phospho-α,β-unsaturated aldehyde (PUA) and 5'-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3'-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5'-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5'-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.
  14. Leukemia. 2020 Dec 09.
      Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KITD816Y AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KITD816Y AML cells in response to chemotherapy. Consistently, combinations of chemotherapy and PI3K or DNA-PK inhibitors synergistically inhibited growth and survival of clonogenic AML cells without substantially inhibiting normal clonogenic bone marrow cells. Moreover, treatment of inv(16)/KITD816Y AML mice with combinations of chemotherapy and PI3K or DNA-PK inhibitors significantly prolonged survival compared to untreated/single-treated mice. Mechanistically, our findings implicate that constitutive activation of PI3K/AKT signaling driven by mutant KIT, and potentially other mutational activators such as FLT3 and RAS, cooperates with chemotherapy-induced DNA-PK-dependent activation of AKT to promote survival, DNA repair, and chemotherapy resistance in AML. Hence, our study provides a rationale to select AML patients exhibiting constitutive PI3K/AKT activation for simultaneous treatment with chemotherapy and inhibitors of DNA-PK and PI3K to improve chemotherapy response and clinical outcome.
  15. Int J Mol Sci. 2020 Dec 03. pii: E9226. [Epub ahead of print]21(23):
      Cancer is the second leading cause of death with tens of millions of people diagnosed with cancer every year around the world. Most radio- and chemotherapies aim to eliminate cancer cells, notably by causing severe damage to the DNA. However, efficient repair of such damage represents a common mechanism of resistance to initially effective cytotoxic agents. Thus, development of new generation anticancer drugs that target DNA repair pathways, and more particularly the base excision repair (BER) pathway that is responsible for removal of damaged bases, is of growing interest. The BER pathway is initiated by a set of enzymes known as DNA glycosylases. Unlike several downstream BER enzymes, DNA glycosylases have so far received little attention and the development of specific inhibitors of these enzymes has been lagging. Yet, dysregulation of DNA glycosylases is also known to play a central role in numerous cancers and at different stages of the disease, and thus inhibiting DNA glycosylases is now considered a valid strategy to eliminate cancer cells. This review provides a detailed overview of the activities of DNA glycosylases in normal and cancer cells, their modes of regulation, and their potential as anticancer drug targets.
    Keywords:  DNA glycosylases; base excision repair; cancer; drug resistance; inhibitors
  16. Mol Genet Metab Rep. 2020 Dec;25 100677
      The PRPS1 gene, located on Xq22.3, encodes phosphoribosyl-pyrophosphate synthetase (PRPS), a key enzyme in de novo purine synthesis. Three clinical phenotypes are associated with loss-of-function PRPS1 variants and decreased PRPS activity: Arts syndrome (OMIM: 301835), Charcot-Marie-Tooth disease type 5 (CMTX5, OMIM: 311070), and nonsyndromic X-linked deafness (DFN2, OMIM: 304500). Hearing loss is present in all cases. CMTX5 patients also show peripheral neuropathy and optic atrophy. Arts syndrome includes developmental delay, intellectual disability, ataxia, and susceptibility to infections, in addition to the above three features. Gain-of-function PRPS1 variants result in PRPS superactivity (OMIM: 300661) with hyperuricemia and gout. We report a 6-year-old boy who presented with marked generalized muscular hypotonia, global developmental delay, lack of speech, trunk instability, exercise intolerance, hypomimic face with open mouth, oropharyngeal dysphagia, dysarthria, and frequent upper respiratory tract infections. However, his nerve conduction velocity, audiologic, and funduscopic investigations were normal. A novel hemizygous variant, c.130A > G p.(Ile44Val), was found in the PRPS1 gene by panel sequencing. PRPS activity in erythrocytes was markedly reduced, confirming the pathogenicity of the variant. Serum uric acid and urinary purine and pyrimidine metabolite levels were normal. In conclusion, we present a novel PRPS1 loss-of-function variant in a patient with some clinical features of Arts syndrome, but lacking a major attribute, hearing loss, which is congenital/early-onset in all other reported Arts syndrome patients. In addition, it is important to acknowledge that normal levels of serum and urinary purine and pyrimidine metabolites do not exclude PRPS1-related disorders.
    Keywords:  Arts syndrome; Autophagy; PRPS1; Purines
  17. Mol Cancer Ther. 2020 Dec 09. pii: molcanther.0316.2020. [Epub ahead of print]
      Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed immunohistochemical analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface co-expression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.
  18. Cancer Biol Med. 2020 Nov 15. 17(4): 805-827
      Viewing cancer as a large, evolving population of heterogeneous cells is a common perspective. Because genomic instability is one of the fundamental features of cancer, this intrinsic tendency of genomic variation leads to striking intratumor heterogeneity and functions during the process of cancer formation, development, metastasis, and relapse. With the increased mutation rate and abundant diversity of the gene pool, this heterogeneity leads to cancer evolution, which is the major obstacle in the clinical treatment of cancer. Cells rely on the integrity of DNA repair machineries to maintain genomic stability, but these machineries often do not function properly in cancer cells. The deficiency of DNA repair could contribute to the generation of cancer genomic instability, and ultimately promote cancer evolution. With the rapid advance of new technologies, such as single-cell sequencing in recent years, we have the opportunity to better understand the specific processes and mechanisms of cancer evolution, and its relationship with DNA repair. Here, we review recent findings on how DNA repair affects cancer evolution, and discuss how these mechanisms provide the basis for critical clinical challenges and therapeutic applications.
    Keywords:  DNA repair; cancer evolution; drug resistance; genomic instability; intratumor heterogeneity
  19. Biochem Pharmacol. 2020 Dec 04. pii: S0006-2952(20)30595-5. [Epub ahead of print] 114359
      Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARP inhibitor induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin- bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/ signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.
    Keywords:  BRCA1 mutant; PARP chromatin-bound; cancer; inflammation
  20. Commun Biol. 2020 Dec 10. 3(1): 751
      Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.
  21. Trends Biochem Sci. 2020 Dec 02. pii: S0968-0004(20)30273-5. [Epub ahead of print]
      Cell cycle checkpoints secure ordered progression from one cell cycle phase to the next. They are important to signal cell stress and DNA lesions and to stop cell cycle progression when severe problems occur. Recent work suggests, however, that the cell cycle control machinery responds in more subtle and sophisticated ways when cells are faced with naturally occurring challenges, such as replication impediments associated with endogenous replication stress. Instead of following a stop and go approach, cells use fine-tuned deceleration and brake release mechanisms under the control of ataxia telangiectasia and Rad3-related protein kinase (ATR) and checkpoint kinase 1 (CHK1) to more flexibly adapt their cell cycle program to changing conditions. We highlight emerging examples of such intrinsic cell cycle checkpoint regulation and discuss their physiological and clinical relevance.
    Keywords:  ATR; CHK1; DNA damage; cell cycle checkpoint signaling; cell cycle control; genome instability; protein degradation; replication stress
  22. Anticancer Res. 2020 Dec;40(12): 6891-6897
      BACKGROUND/AIM: Cellular senescence is an important tumor-suppressive mechanism that arrests the cell cycle of damaged cells after diverse stresses. This study aimed to elucidate the role of mitochondrial glutamine (Gln) metabolism in senescence cell-fate decision after DNA damage.MATERIALS AND METHODS: β-galactosidase staining was used to determine senescence induction. The mechanistic target of rapamycin (mTOR) activity and p21 expression were examined by western blot. Cell proliferation and clonogenic growth were evaluated.
    RESULTS: Inhibition of mitochondrial Gln metabolism suppressed DNA damage-induced senescence, whereas increased Gln anaplerosis resulted in a profound induction of senescence. Mechanistically, Gln anaplerosis mediated senescence induction by activating mTOR signaling upon DNA damage. Importantly, enhancing Gln anaplerosis could reduce the emergence of proliferative subpopulations of cancer cells after exposure to non-lethal doses of chemotherapeutic agents.
    CONCLUSION: Mitochondrial Gln metabolism is an important regulator of DNA damage-induced senescence, which may be used for developing effective therapeutic approaches.
    Keywords:  DNA damage; Senescence; glutamine metabolism; mTOR
  23. PLoS Genet. 2020 Dec 10. 16(12): e1009260
      TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R-loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R-loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.
  24. Elife. 2020 Dec 08. pii: e61920. [Epub ahead of print]9
      Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.
    Keywords:  chromosomes; gene expression; molecular biophysics; structural biology; xenopus
  25. Mol Cell. 2020 Nov 27. pii: S1097-2765(20)30788-7. [Epub ahead of print]
      Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.
    Keywords:  MRX/N; Mediator; SMC complexes; chromosomal interaction domains; chromosome folding; chromosome organization; coding and non-coding transcription control; nuclear pore
  26. Oncogenesis. 2020 Dec 07. 9(12): 104
      Human HLTF participates in the lesion-bypass mechanism through the fork reversal structure, known as template switching of post-replication repair. However, the mechanism by which HLTF promotes the replication progression and fork stability of damaged forks remains unclear. Here, we identify a novel protein-protein interaction between HLTF and PARP1. The depletion of HLTF and PARP1 increases chromosome breaks, further reduces the length of replication tracks, and concomitantly increases the number of stalled forks after methyl methanesulfonate treatment according to a DNA fiber analysis. The progression of replication also depends on BARD1 in the presence of MMS treatment. By combining 5-ethynyl-2'-deoxyuridine with a proximity ligation assay, we revealed that the HLTF, PARP1, and BRCA1/BARD1/RAD51 proteins were initially recruited to damaged forks. However, prolonged stalling of damaged forks results in fork collapse. HLTF and PCNA dissociate from the collapsed forks, with increased accumulation of PARP1 and BRCA1/BARD1/RAD51 at the collapsed forks. Our results reveal that HLTF together with PARP1 and BARD1 participates in the stabilization of damaged forks, and the PARP1-BARD1 interaction is further involved in the repair of collapse forks.
  27. Mol Cancer Ther. 2020 Dec 08. pii: molcanther.0567.2020. [Epub ahead of print]
      LKB1-inactivated tumors are vulnerable to the disruption of pyrimidine metabolism, and leflunomide emerges as a therapeutic candidate because its active metabolite, A77-1726, inhibits dihydroorotate dehydrogenase, which is essential for de novo pyrimidine biosynthesis. However, it is unclear whether leflunomide inhibits LKB1-inactivated tumors in vivo, and whether its inhibitory effect on the immune system will promote tumor growth. Here, we carried out a comprehensive analysis of leflunomide treatment in various LKB1-inactivated murine xenograft, PDX, and genetically engineered mouse models. We also generated a mouse-tumor derived cancer cell line, WRJ388, that could metastasize to the lung within a month after subcutaneous implantation in all animals. This model was used to assess the ability of leflunomide to control distant metastasis. Leflunomide treatment shrank a HeLa xenograft and attenuated the growth of an H460 xenograft, a PDX, and lung adenocarcinoma in the immune-competent GEMM. Interestingly, leflunomide suppressed tumor growth through at least three different mechanisms. It caused apoptosis in HeLa cells, induced G1 cell cycle arrest in H460 cells, and promoted S-phase cell cycle arrest in WRJ388 cells. Finally, leflunomide treatment prevented lung metastasis in 78% of the animals in our novel lung cancer metastasis model. In combination, these results demonstrated that leflunomide utilizes different pathways to suppress the growth of LKB1-inactivated tumors, and it also prevents cancer metastasis at distant sites. Therefore, leflunomide should be evaluated as a therapeutic agent for tumors with LKB1-inactivation.