bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2020‒12‒20
27 papers selected by
Sean Rudd
Karolinska Institutet

  1. Mol Cell. 2020 Dec 15. pii: S1097-2765(20)30898-4. [Epub ahead of print]
      Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
    Keywords:  ALC1; BRCAs; DNA damage repair; DNA gycosylases; PARPs; base excsion repair; chromatin remodeler; homologous recombination defieciency; poly(ADP)-ribosylation; synthetic lethality
  2. Cancer Cell. 2020 Dec 03. pii: S1535-6108(20)30596-1. [Epub ahead of print]
      Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.
    Keywords:  DNA breaks; MLH1; RPA exhaustion; Rad51; cGAS-STING; chromosome instability; cytosolic DNA; exonuclease 1; mismatch repair
  3. Cancer Cell. 2020 Nov 28. pii: S1535-6108(20)30598-5. [Epub ahead of print]
      Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-β in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.
    Keywords:  DNA sensing; MLH1; MSI; STING; T cell infiltration; cGAS; cancer; checkpoint blockade; cytosolic DNA; mismatch repair
  4. Cell Metab. 2020 Dec 10. pii: S1550-4131(20)30657-4. [Epub ahead of print]
      Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.
    Keywords:  SHMT; cancer metabolism; folate cycle; in vivo; isotope tracing; metabolomics; mitochondria; one-carbon flux; physiologic medium; reduced folate carrier; serine hydroxymethyltransferase
  5. Nucleic Acids Res. 2020 Dec 16. pii: gkaa1201. [Epub ahead of print]
      Many APOBEC cytidine deaminase members are known to induce 'off-target' cytidine deaminations in 5'TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.
  6. Drug Discov Today. 2020 Dec 11. pii: S1359-6446(20)30524-9. [Epub ahead of print]
      Folate-mediated one-carbon metabolism (FOCM) supports vital events for the growth and survival of proliferating cells. Nucleotide synthesis and DNA methylation are the biochemical bases of cancers that are highly dependent on FOCM. Recent studies revealed that FOCM is connected with redox homeostasis and epigenetics in cancer. Furthermore, folate-metabolizing enzymes, such as serine hydroxymethyltransferase 2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), are associated with the development of cancers, including breast cancer, highlighting their potential application in tumor-targeted therapy. Therefore, targeting metabolizing enzymes, especially SHMT2 and MTHFD2, provides a novel strategy for cancer treatment. In this review, we outline current understanding of the functions of SHMT2 and MTHFD2, discussing their expression, potential functions, and regulatory mechanism in cancers. Furthermore, we discuss examples of inhibitors of SHMT2 and MTHFD2.
  7. Hepatology. 2020 Dec 17.
      Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide; yet, patients with HCC have limited treatment options. There is an urgent need to identify new drug targets that specifically inhibit the growth of HCC cells. Here, we used a newly-engineered CRISPR library targeting ~2,000 druggable genes to perform a high throughput screen, and identified adenylosuccinate lyase (ADSL) - a key enzyme involved in the de novo purine synthesis pathway - as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting adenosine monophosphate (AMP) production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest, not by inducing DNA damage, but by impairing mitochondrial function. Using HCC patient data, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. In conclusion, our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for HCC patients.
    Keywords:  ATP; CRISPR; Cell Cycle; Hepatocellular Carcinoma; Mitochondria
  8. Nucleic Acids Res. 2020 Dec 17. pii: gkaa1184. [Epub ahead of print]
      RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
  9. ACS Chem Biol. 2020 Dec 17.
      DNA glycosylases involved in the first step of the DNA base excision repair pathway are promising targets in cancer therapy. There is evidence that reduction of their activities may enhance cell killing in malignant tumors. Recently, two tetrahydroquinoline compounds named SU0268 and SU0383 were reported to inhibit OGG1 for the excision of 8-hydroxyguanine. This DNA repair protein is one of the major cellular enzymes responsible for excision of a number of oxidatively induced lesions from DNA. In this work, we used gas chromatography-tandem mass spectrometry with isotope-dilution to measure the excision of not only 8-hydroxyguanine but also that of the other major substrate of OGG1, i.e., 2,6-diamino-4-hydroxy-5-formamidopyrimidine, using genomic DNA with multiple purine- and pyrimidine-derived lesions. The excision of a minor substrate 4,6-diamino-5-formamidopyrimidine was also measured. Both SU0268 and SU0383 efficiently inhibited OGG1 activity for these three lesions, with the former being more potent than the latter. Dependence of inhibition on concentrations of SU0268 and SU0383 from 0.05 μmol/L to 10 μmol/L was also demonstrated. The approach used in this work may be applied to the investigation of OGG1 inhibition by SU0268 and SU0383 and other small molecule inhibitors in further studies including cellular and animal models of disease.
  10. ChemMedChem. 2020 Dec 13.
      Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low-fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance efficacy of first-line chemotherapy, with the majority of reported inhibitors targeting protein-protein interactions (PPIs) of the Rev1 C-terminal domain (Rev1-CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1-CT PPIs with Rev1 interacting regions (RIRs) of TLS polymerases. To explore structure-activity relationships for this scaffold, here we developed a protocol for co-crystallization of compounds that target the RIR-binding site on Rev1-CT with a triple Rev1-CT/Rev7R124A/Rev3-RBM1 complex, and solved an X-ray crystal structure of Rev1-CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1-CT. We synthesized 8 additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1-CT by microscale thermophoresis (MST). Several second-generation PAP derivatives showed over an order of magnitude improved affinity for Rev1-CT, validating structure-based assumptions that went into the compound design.
    Keywords:  cancer chemotherapy; crystallography; protein-protein interactions; structure activity relationships; translesion synthesis inhibitors
  11. Trends Cell Biol. 2020 Dec 11. pii: S0962-8924(20)30232-4. [Epub ahead of print]
      In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.
    Keywords:  DNA interstrand crosslink repair; DNA-protein crosslink repair; E3 ubiquitin ligase; mitotic DNA replication; replication termination
  12. Exp Mol Med. 2020 Dec 18.
      Eukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).
  13. Nucleic Acids Res. 2020 Dec 16. pii: gkaa1202. [Epub ahead of print]
      DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.
  14. Front Cell Dev Biol. 2020 ;8 595687
      Human telomeres as well as more than 40% of human genes near the promoter regions have been found to contain the sequence that may form a G-quadruplex structure. Other non-canonical DNA structures comprising bulges, hairpins, or bubbles may have a functionally important role during transcription, replication, or recombination. The guanine-rich regions of DNA are hotspots of oxidation that forms 7,8-dihydro-8-oxoguanine, thymine glycol, and abasic sites: the lesions that are handled by the base excision repair pathway. Nonetheless, the features of DNA repair processes in non-canonical DNA structures are still poorly understood. Therefore, in this work, a comparative analysis of the efficiency of the removal of a damaged nucleotide from various G-quadruplexes and bulged structures was performed using endonuclease VIII-like 1 (NEIL1), human 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III (NTH1), and prokaryotic formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei). All the tested enzymes were able to cleave damage-containing bulged DNA structures, indicating their important role in the repair process when single-stranded DNA and intermediate non-B-form structures such as bubbles and bulges are formed. Nevertheless, our results suggest that the ability to cleave damaged quadruplexes is an intrinsic feature of members of the H2tH structural family, suggesting that these enzymes can participate in the modulation of processes controlled by the formation of quadruplex structures in genomic DNA.
    Keywords:  DNA bulge; DNA glycosylase; G-quadruplex; base excision repair; fluorescence; pre-steady-state kinetics
  15. J Exp Med. 2021 Mar 01. pii: e20200622. [Epub ahead of print]218(3):
      ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.
  16. Sci Rep. 2020 Dec 16. 10(1): 22015
      Combination chemotherapy is still of great importance as part of the standard clinical care for patients with HER2 positive breast cancer. As an attractive component, gold nanoparticles (AuNPs) have been extensively studied as biosafety nanomaterials, but they are rarely explored as drug nanocarriers for targeted co-delivery of multiple chemotherapeutics. Herein, a novel affibody-DNA hybrid strands modified AuNPs were fabricated for co-loading nucleoside analogue (5-fluorodeoxyuridine, FUdR) and anthracycline (doxorubicin, Dox). FUdRs were integrated into DNA hybrid strands decorated on AuNPs by DNA solid phase synthesis, and Dox molecules were intercalated into their duplex regions. Affibody molecules coupled to the DNA hybrid strands were distributed the surface of AuNPs, giving them targeting for HER2. The new dual-drug-containing affibody-DNA-AuNPs (Dox@affi-F/AuNPs) owned compact and stable spherical nanostructures, and precise drug loading. Cytotoxicity tests demonstrated that these nanoparticles caused a higher inhibition in HER2 overexpressing breast cancer cells, and showed better synergistic antitumor activity than simple mixture of the two drugs. The related mechanistic studies proved that Dox@affi-F/AuNPs achieved a remarkable combined antitumor activity of Dox and FUdR by promoting more cells to enter apoptosis pathway. Our work provided a nanomedicine platform for targeted co-delivery of nucleoside analog therapeutics and anthracycline anticancer drugs to achieve synergistic treatment of HER2+ cancer.
  17. Nucleic Acids Res. 2020 Dec 16. pii: gkaa1188. [Epub ahead of print]
      The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.
  18. PLoS One. 2020 ;15(12): e0237759
      DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in "clustered" oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in "real time" by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.
  19. Clin Cancer Res. 2020 Dec 17. pii: clincanres.2483.2020. [Epub ahead of print]
      PURPOSE: Alterations in DNA damage repair (DDR) pathway genes occur in 20-25% of men with metastatic castration-resistant prostate cancer (mCRPC). Although poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) have been shown to benefit men with mCRPC harboring DDR defects due to mutations in BRCA1/2 and ATM, additional treatments are necessary because the effects are not durable.EXPERIMENTAL DESIGN: We performed transcriptomic analysis of publicly available mCRPC cases, comparing BRCA2-null to BRCA2 wild type. We generated BRCA2-null prostate cancer cells using CRISPR/Cas9 and treated these cells with PARPi and SRC inhibitors. We also assessed the antiproliferative effects of combination treatment in 3D prostate cancer organoids.
    RESULTS: We observed significant enrichment of the SRC signaling pathway in BRCA2-altered mCRPC. BRCA2-null prostate cancer cell lines had increased SRC phosphorylation and higher sensitivity to SRC inhibitors (eg, dasatinib, bosutinib, and saracatinib) relative to wild-type cells. Combination treatment with PARPi and SRC inhibitors was antiproliferative and had a synergistic effect in BRCA2-null prostate cancer cells, mCRPC organoids, and Trp53/Rb1-null prostate cancer cells. Inhibition of SRC signaling by dasatinib augmented DNA damage in BRCA2-null prostate cancer cells. Moreover, SRC knockdown increased PARPi sensitivity in BRCA2-null prostate cancer cells.
    CONCLUSIONS: This work suggests that SRC activation may be a potential mechanism of PARPi resistance and that treatment with SRC inhibitors may overcome this resistance. Our preclinical study demonstrates that combining PARPi and SRC inhibitors may be a promising therapeutic strategy for patients with BRCA2-null mCRPC.
  20. Curr Biol. 2020 Dec 08. pii: S0960-9822(20)31748-6. [Epub ahead of print]
      Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migration-induced DNA damage remain incompletely understood. Although in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that, in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear-envelope-rupture-induced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication stress, possibly by increasing replication fork stalling, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells but could also cause DNA damage in non-migrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.
    Keywords:  DNA damage; cancer; cell compression; confined migration; metastasis; nuclear deformation; nuclear envelope rupture; nuclear mechanobiology; replication stress
  21. J Biol Chem. 2020 Dec 11. pii: jbc.RA120.016489. [Epub ahead of print]
      Magnesium ions play a critical role in catalysis by many enzymes and they contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ions in each step in catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. Increasing Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold by largely increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV RT binds only two metal ions prior to incorporation of a correct base-pair. MD simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. MD simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions the specificity of DNA polymerases.
    Keywords:  DNA enzyme; DNA replication; conformational change; enzyme specificity; global data fitting; induced-fit; magnesium; molecular dynamics; nucleic acid enzymology; translocation; two-metal ion
  22. Cancer Lett. 2020 Dec 11. pii: S0304-3835(20)30662-5. [Epub ahead of print]
      Fanconi anemia (FA) is a chromosomal instability disorder of bone marrow associated with aplastic anemia, congenital abnormalities and a high risk of malignancies. The identification of more than two dozen FA genes has revealed a plethora of interacting proteins that are mainly involved in repair of DNA interstrand crosslinks (ICLs). Other important findings associated with FA are inflammation, oxidative stress response, mitochondrial dysfunction and mitophagy. In this work, we performed quantitative proteomic and metabolomic analyses on defective FA cells and identified a number of metabolic abnormalities associated with cancer. In particular, an increased de novo purine biosynthesis, a high concentration of fumarate, and an accumulation of purinosomal clusters were found. This was in parallel with decreased OXPHOS and altered glycolysis. On the whole, our results indicate an association between the need for nitrogenous bases upon impaired DDR in FA cells with a subsequent increase in purine metabolism and a potential role in oncogenesis.
    Keywords:  Cancer predisposition; Fanconi anemia; Fumarate; Metabolic reprogramming; Purinosome; de novo purine biosynthesis
  23. Nucleic Acids Res. 2020 Dec 11. pii: gkaa1172. [Epub ahead of print]
      G-Quadruplexes are non-B form DNA structures present at regulatory regions in the genome, such as promoters of proto-oncogenes and telomeres. The prominence in such sites suggests G-quadruplexes serve an important regulatory role in the cell. Indeed, oxidized G-quadruplexes found at regulatory sites are regarded as epigenetic elements and are associated with an interlinking of DNA repair and transcription. PARP-1 binds damaged DNA and non-B form DNA, where it covalently modifies repair enzymes or chromatin-associated proteins respectively with poly(ADP-ribose) (PAR). PAR serves as a signal in regulation of transcription, chromatin remodeling, and DNA repair. PARP-1 is known to bind G-quadruplexes with stimulation of enzymatic activity. We show that PARP-1 binds several G-quadruplex structures with nanomolar affinities, but only a subset promote PARP-1 activity. The G-quadruplex forming sequence found in the proto-oncogene c-KIT promoter stimulates enzymatic activity of PARP-1. The loop-forming characteristics of the c-KIT G-quadruplex sequence regulate PARP-1 catalytic activity, whereas eliminating these loop features reduces PARP-1 activity. Oxidized G-quadruplexes that have been suggested to form unique, looped structures stimulate PARP-1 activity. Our results support a functional interaction between PARP-1 and G-quadruplexes. PARP-1 enzymatic activation by G-quadruplexes is dependent on the loop features and the presence of oxidative damage.
  24. Front Pharmacol. 2020 ;11 585876
      Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are ectoenzymes that play an important role in the hydrolysis of nucleoside triphosphate and diphosphate to nucleoside monophosphate. NTPDase1, -2, -3 and -8 are the membrane bound members of this enzyme family that are responsible for regulating the levels of nucleotides in extracellular environment. However, the pathophysiological functions of these enzymes are not fully understood due to lack of potent and selective NTPDase inhibitors. Herein, a series of oxoindolin hydrazine carbothioamide derivatives is synthesized and screened for NTPDase inhibitory activity. Four compounds were identified as selective inhibitors of h-NTPDase1 having IC50 values in lower micromolar range, these include compounds 8b (IC50 = 0.29 ± 0.02 µM), 8e (IC50 = 0.15 ± 0.009 µM), 8f (IC50 = 0.24 ± 0.01 µM) and 8l (IC50 = 0.30 ± 0.03 µM). Similarly, compound 8k (IC50 = 0.16 ± 0.01 µM) was found to be a selective h-NTPDase2 inhibitor. In case of h-NTPDase3, most potent inhibitors were compounds 8c (IC50 = 0.19 ± 0.02 µM) and 8m (IC50 = 0.38 ± 0.03 µM). Since NTPDase3 has been reported to be associated with the regulation of insulin secretion, we evaluated our synthesized NTPDase3 inhibitors for their ability to stimulate insulin secretion in isolated mice islets. Promising results were obtained showing that compound 8m potently stimulated insulin secretion without affecting the NTPDase3 gene expression. Molecular docking studies of the most potent compounds were also carried out to rationalize binding site interactions. Hence, these compounds are useful tools to study the role of NTPDase3 in insulin secretion.
    Keywords:  carbothioamide; ectonucleotidases; molecular docking; nucleoside triphosphate diphosphohydrolase-3; oxoindolin hydrazine
  25. Nature. 2020 Dec 16.
      Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.
  26. Mol Cell. 2020 Dec 17. pii: S1097-2765(20)30840-6. [Epub ahead of print]80(6): 1013-1024.e6
      Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.
    Keywords:  DNA damage; Fanconi anemia; bone marrow failure; differentiation; formaldehyde; hematopoiesis; transcription reprogramming
  27. Curr Opin Cell Biol. 2020 Dec 10. pii: S0955-0674(20)30151-4. [Epub ahead of print]70 27-36
      Subversion of genome integrity fuels cellular adaptation and is a prerequisite for organismal evolution, yet genomic lesions are also the harmful driving force of cancer and other age-related human diseases. Genome integrity maintenance is inherently linked to genome organization and nuclear architecture, which are substantially remodeled during the cell cycle. Here we discuss recent findings on how actively dividing cells cope with endogenous genomic lesions that occur frequently at repetitive, heterochromatic, and late replicating regions as byproducts of genome duplication. We discuss how such lesions, rather than being resolved immediately when they occur, are dealt with in subsequent cell cycle phases, and even after mitotic cell division, and how this in turn affects genome organization, stability, and function.
    Keywords:  ALT; Cell cycle control; Chromatin compartments; Genome stability; Liquid–liquid phase separation; MiDAS; Mitosis; Telomere maintenance