bims-numges 2022-07-17 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–07–17
twenty-six papers selected by
Sean Rudd, Karolinska Institutet

Stalled replication fork protection limits cGAS-STING and P-body-dependent innate immune signalling.
Enhanced polymerase activity permits efficient synthesis by cancer-associated DNA polymerase ϵ variants at low dNTP levels.
BRCA1-dependent and independent recruitment of PALB2-BRCA2-RAD51 in the DNA damage response and cancer.
A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells.
Reconstitution of a telomeric replicon organized by CST.
Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction.
Structures of the human CST-Polα-primase complex bound to telomere templates.
Sen1 is a key regulator of transcription-driven conflicts.
Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.
Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine.
DNA repair as a shared hallmark in cancer and ageing.
Next-Generation Sequencing-Based Analysis of the Roles of DNA Polymerases ν and θ in the Replicative Bypass of 8-Oxo-7,8-dihydroguanine in Human Cells.
Enhancing Repair of Oxidative DNA Damage with Small-Molecule Activators of MTH1.
Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease.
Guiding ATR and PARP inhibitor combinationswith chemogenomic screens.
Molecular crowding facilitates bundling of IMPDH polymers and cytoophidium formation.
A non-transcriptional function of Yap regulates the DNA replication program.
Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.
A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase.
Polβ modulates the expression of type I interferon via STING pathway.
CDK7 inhibition synergizes with topoisomerase I inhibition in small cell lung cancer cells by inducing ubiquitin-mediated proteolysis of RNA polymerase II.
Resolution of R-loops by topoisomerase III-β (TOP3B) in coordination with the DEAD-box helicase DDX5.
Discovery of CN0 as a novel proteolysis-targeting chimera (PROTAC) degrader of PARP1 that can activate the cGAS/STING immunity pathway combined with daunorubicin.
MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state.
Nrf2 overexpression increases the resistance of acute myeloid leukemia to cytarabine by inhibiting replication factor C4.
Bacteria deplete deoxynucleotides to defend against bacteriophage infection.

Nat Cell Biol. 2022 Jul;24(7): 1154-1164

Stalled replication fork protection limits cGAS-STING and P-body-dependent innate immune signalling.

Ahmed Emam, Xiao Wu, Shengfeng Xu, Longqiang Wang, Shichang Liu, Bin Wang.

Protection of stalled replication forks is crucial for cells to respond to replication stress and maintain genome stability. Genome instability and replication stress have been linked to immune activation. Here we show that Abro1 and FANCD2 protect replication forks, which is linked with the restriction of innate immune responses. We reveal that stalled replication fork degradation induced by Abro1 or FANCD2 deficiency leads to accumulation of cytosolic single-stranded DNA and activation of a cGAS-STING-dependent innate immune response that is dependent on DNA2 nuclease. We further show that the increased cytosolic single-stranded DNA contains ribosomal DNA that can bind to cGAS. In addition, Abro1 and FANCD2 limit the formation of replication stress-induced P-bodies, and P-bodies are capable of modulating activation of the innate immune response after prolonged replication stress. Our study demonstrates a connection between replication stress and activation of the innate immune response that may be targeted for therapeutic purpose.

DOI: https://doi.org/10.1038/s41556-022-00950-8
Nucleic Acids Res. 2022 Jul 13. pii: gkac602. [Epub ahead of print]

Enhanced polymerase activity permits efficient synthesis by cancer-associated DNA polymerase ϵ variants at low dNTP levels.

Stephanie R Barbari, Annette K Beach, Joel G Markgren, Vimal Parkash, Elizabeth A Moore, Erik Johansson, Polina V Shcherbakova.

Amino acid substitutions in the exonuclease domain of DNA polymerase ϵ (Polϵ) cause ultramutated tumors. Studies in model organisms suggested pathogenic mechanisms distinct from a simple loss of exonuclease. These mechanisms remain unclear for most recurrent Polϵ mutations. Particularly, the highly prevalent V411L variant remained a long-standing puzzle with no detectable mutator effect in yeast despite the unequivocal association with ultramutation in cancers. Using purified four-subunit yeast Polϵ, we assessed the consequences of substitutions mimicking human V411L, S459F, F367S, L424V and D275V. While the effects on exonuclease activity vary widely, all common cancer-associated variants have increased DNA polymerase activity. Notably, the analog of Polϵ-V411L is among the strongest polymerases, and structural analysis suggests defective polymerase-to-exonuclease site switching. We further show that the V411L analog produces a robust mutator phenotype in strains that lack mismatch repair, indicating a high rate of replication errors. Lastly, unlike wild-type and exonuclease-dead Polϵ, hyperactive variants efficiently synthesize DNA at low dNTP concentrations. We propose that this characteristic could promote cancer cell survival and preferential participation of mutator polymerases in replication during metabolic stress. Our results support the notion that polymerase fitness, rather than low fidelity alone, is an important determinant of variant pathogenicity.

DOI: https://doi.org/10.1093/nar/gkac602
Cancer Res. 2022 Jul 12. pii: can.22.1535. [Epub ahead of print]

BRCA1-dependent and independent recruitment of PALB2-BRCA2-RAD51 in the DNA damage response and cancer.

Tzeh Keong Foo, Bing Xia.

The BRCA1-PALB2-BRCA2 axis plays essential roles in the cellular response to DNA double strand breaks (DSB), maintenance of genome integrity, and suppression of cancer development. Upon DNA damage, BRCA1 is recruited to DSBs, where it facilitates end resection and recruits PALB2 and its associated BRCA2 to load the central recombination enzyme RAD51 to initiate homologous recombination (HR) repair. In recent years, several BRCA1-independent mechanisms of PALB2 recruitment have also been reported. Collectively, these available data illustrate a series of hierarchical, context-dependent, and cooperating mechanisms of PALB2 recruitment that is critical for HR and therapy response either in the presence or absence of BRCA1. Here, we review these BRCA1-dependent and independent mechanisms and their importance in DSB repair, cancer development, and therapy. As BRCA1-mutant cancer cells regain HR function, for which PALB2 is generally required, and become resistant to targeted therapies, such as PARP inhibitors, targeting BRCA1-independent mechanisms of PALB2 recruitment represents a potential new avenue to improve treatment of BRCA1-mutant tumors.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-1535
Bio Protoc. 2022 May 20. 12(10): e4413

A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells.

Bo-Ruei Chen, Jessica K Tyler, Barry P Sleckman.

  DNA double strand breaks (DSBs) constantly arise in cells during normal cellular processes or upon exposure to genotoxic agents, and are repaired mostly by homologous recombination (HR) and non-homologous end joining (NHEJ). One key determinant of DNA DSB repair pathway choice is the processing of broken DNA ends to generate single strand DNA (ssDNA) overhangs, a process termed DNA resection. The generation of ssDNA overhangs commits DSB repair through HR and inhibits NHEJ. Therefore, DNA resection must be carefully regulated to avoid mis-repaired or persistent DSBs. Accordingly, many approaches have been developed to monitor ssDNA generation in cells to investigate genes and pathways that regulate DNA resection. Here we describe a flow cytometric approach measuring the levels of replication protein A (RPA) complex, a high affinity ssDNA binding complex composed of three subunits (RPA70, RPA32, and RPA14 in mammals), on chromatin after DNA DSB induction to assay DNA resection. This flow cytometric assay requires only conventional flow cytometers and can easily be scaled up to analyze a large number of samples or even for genetic screens of pooled mutants on a genome-wide scale. We adopt this assay in G0- and G1- phase synchronized cells where DNA resection needs to be kept in check to allow normal NHEJ.

Keywords:  DNA DSB; Flow cytometry; HR; NHEJ; RPA; Resection; ssDNA

DOI:  https://doi.org/10.21769/BioProtoc.4413
Nature. 2022 Jul 13.

Reconstitution of a telomeric replicon organized by CST.

Arthur J Zaug, Karen J Goodrich, Jessica J Song, Ashley E Sullivan, Thomas R Cech.

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6-9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3' overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα-primase to telomeric ssDNA4,12,13 but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

DOI: https://doi.org/10.1038/s41586-022-04930-8
Nucleic Acids Res. 2022 Jul 12. pii: gkac588. [Epub ahead of print]

Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction.

Matthew Day, Antony W Oliver, Laurence H Pearl.

The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5'-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5' DNA end and fixes the orientation of 9-1-1 on the ssDNA.

DOI: https://doi.org/10.1093/nar/gkac588
Nature. 2022 Jul 13.

Structures of the human CST-Polα-primase complex bound to telomere templates.

Qixiang He, Xiuhua Lin, Bianca L Chavez, Sourav Agrawal, Benjamin L Lusk, Ci Ji Lim.

The mammalian DNA polymerase-alpha/primase (pol-α/primase) is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The underlying physical mechanism of how pol-α/primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here, we show that CST, a single-stranded DNA-binding accessory protein complex of pol-α/primase, physically sets up the enzyme for efficient primer synthesis. Cryo-electron microscopy structures of CST-pol-α/primase preinitiation complex (PIC) bound to various types of telomere overhang reveal template-bound CST partitions the DNA and RNA catalytic centers of pol-α/primase into two separate domains and effectively arrange them in RNA-DNA synthesis order. The PIC architecture provides a single solution for the multiple structural needs for pol-α/primase RNA-DNA primer synthesis. Multiple insights into CST template-binding specificity, template requirement for CST-pol-α/primase PIC assembly, and activation are also revealed in this study.

DOI: https://doi.org/10.1038/s41586-022-05040-1
Mol Cell. 2022 Jul 08. pii: S1097-2765(22)00604-9. [Epub ahead of print]

Sen1 is a key regulator of transcription-driven conflicts.

Umberto Aiello, Drice Challal, Griselda Wentzinger, Armelle Lengronne, Rowin Appanah, Philippe Pasero, Benoit Palancade, Domenico Libri.

  Cellular homeostasis requires the coordination of several machineries concurrently engaged in the DNA. Wide-spread transcription can interfere with other processes, and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase not only terminates non-coding transcription but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability, but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that, under physiological conditions, Sen1 removes RNA polymerase II at TRCs within genes and the rDNA and at sites of transcription-transcription conflicts, thus qualifying as a "key regulator of conflicts." We demonstrate that genomic stability is affected by Sen1 mutation only when in addition to its role at the replisome, the termination of non-coding transcription or R-loop removal are additionally compromised.

Keywords:  H-CRAC; R-loops; RNase H; Sen1; TRCs; genome stability; non-coding transcription; replication; transcription; transcription-replication conflicts

DOI:  https://doi.org/10.1016/j.molcel.2022.06.021
Elife. 2022 Jul 13. pii: e77469. [Epub ahead of print]11

Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.

Fengwei Zheng, Roxana E Georgescu, Nina Y Yao, Huilin Li, Michael E O'Donnell.

  RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases d and e. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3' and 5' ends are present at a ssDNA gap, we propose that the 5' site facilitates RFC's PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5'-DNA site in lagging strand DNA synthesis.

Keywords:  S. cerevisiae; biochemistry; chemical biology

DOI:  https://doi.org/10.7554/eLife.77469
Nucleic Acids Res. 2022 Jul 12. pii: gkac545. [Epub ahead of print]

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine.

Aldo Spanjaard, Ronak Shah, Daniël de Groot, Olimpia Alessandra Buoninfante, Ben Morris, Cor Lieftink, Colin Pritchard, Lisa M Zürcher, Shirley Ormel, Joyce J I Catsman, Renske de Korte-Grimmerink, Bjørn Siteur, Natalie Proost, Terry Boadum, Marieke van de Ven, Ji-Ying Song, Maaike Kreft, Paul C M van den Berk, Roderick L Beijersbergen, Heinz Jacobs.

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.

DOI: https://doi.org/10.1093/nar/gkac545
Mol Oncol. 2022 Jul 14.

DNA repair as a shared hallmark in cancer and ageing.

Thomas L Clarke, Raul Mostoslavsky.

  Increasing evidence demonstrates that DNA damage and genome instability play a crucial role in ageing. Mammalian cells have developed a wide range of complex and well-orchestrated DNA repair pathways to respond to and resolve the many different types of DNA lesions that occur from exogenous and endogenous sources. Defects in these repair pathways lead to accelerated or premature ageing syndromes and increase the likelihood of cancer development. Understanding the fundamental mechanisms of DNA repair will help develop novel strategies to treat ageing-related diseases. Here, we revisit the processes involved in DNA damage repair and how these can contribute to diseases, including aging and cancer. We also review recent mechanistic insights into DNA repair and discuss how these insights are being used to develop novel therapeutic strategies for treating human disease. We discuss the use of PARP inhibitors in the clinic for the treatment of breast and ovarian cancer and the challenges associated with acquired drug resistance. Finally, we discuss how DNA repair pathway-targeted therapeutics are moving beyond PARP inhibition in the search for ever more innovative and efficacious cancer therapies.

Keywords:  DNA damage; ageing; cancer; genome instability; therapeutics

DOI:  https://doi.org/10.1002/1878-0261.13285
ACS Chem Biol. 2022 Jul 10.

Next-Generation Sequencing-Based Analysis of the Roles of DNA Polymerases ν and θ in the Replicative Bypass of 8-Oxo-7,8-dihydroguanine in Human Cells.

Yini Liu, Xiaowen Zhu, Ziyu Wang, Xiaoxia Dai, Changjun You.

DNA polymerase (Pol) ν and Pol θ are two specialized A-family DNA polymerases that function in the translesion synthesis of certain DNA lesions. However, the biological functions of human Pols ν and θ in cellular replicative bypass of 8-oxo-7,8-dihydroguanine (8-oxoG), an important carcinogenesis-related biomarker of oxidative DNA damage, remain unclear. Herein, we showed that depletion of Pols ν and θ in human cells could cause an elevated hypersensitivity to oxidative stress induced by hydrogen peroxide. Using next-generation sequencing-based lesion bypass and mutagenesis assay, we further demonstrated that Pols ν and θ had important roles in promoting translesion synthesis of 8-oxoG in human cells. We also found that the depletion of Pol ν, but not Pol θ, caused a substantial reduction in G → T mutation frequency for 8-oxoG. These findings provided novel insights into the involvement of A-family DNA polymerases in oxidative DNA damage response.

DOI: https://doi.org/10.1021/acschembio.2c00415
ACS Chem Biol. 2022 Jul 13.

Enhancing Repair of Oxidative DNA Damage with Small-Molecule Activators of MTH1.

Yujeong Lee, Yoshiyuki Onishi, Lisa McPherson, Anna M Kietrys, Marian Hebenbrock, Yong Woong Jun, Ishani Das, Shanthi Adimoolam, Debin Ji, Michael G Mohsen, James M Ford, Eric T Kool.

Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. Here, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in the treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-2'-deoxyguanosine (8-oxo-dG)TP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogues of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 μM and 410 ± 60% at 5 μM. The compounds are found to increase the activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks.

DOI: https://doi.org/10.1021/acschembio.2c00038
Biochim Biophys Acta Gen Subj. 2022 Jul 06. pii: S0304-4165(22)00116-7. [Epub ahead of print] 130198

Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease.

Artemiy S Bakman, Alexander A Ishchenko, Murat Saparbaev, Olga S Fedorova, Nikita A Kuznetsov.

  Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5' side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3'-5'-exonuclease, 3'-phosphodiesterase, and 3'-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre-steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding-site residue, Arg181, was analyzed too; it changed its conformation when enzyme-substrate and enzyme-product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction.

Keywords:  Active site; Apurinic/apyrimidinic endonuclease; Conformational change; DNA repair; DNA-protein interaction; Fluorescence; Pre-steady-state kinetics; Substrate recognition

DOI:  https://doi.org/10.1016/j.bbagen.2022.130198
Cell Rep. 2022 Jul 12. pii: S2211-1247(22)00879-8. [Epub ahead of print]40(2): 111081

Guiding ATR and PARP inhibitor combinationswith chemogenomic screens.

Michal Zimmermann, Cynthia Bernier, Beatrice Kaiser, Sara Fournier, Li Li, Jessica Desjardins, Alexander Skeldon, Victoria Rimkunas, Artur Veloso, Jordan T F Young, Anne Roulston, Michael Zinda.

  Combinations of ataxia telangiectasia- and Rad3-related kinase inhibitors (ATRis) and poly(ADP-ribose) polymerase inhibitors (PARPis) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this, we performed a genome-wide CRISPR-Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations, or the "alternative lengthening of telomeres" telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable in vivo dosing schedule. We present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.

Keywords:  ATR inhibitor; CP: Cancer; CRISPR screen; PARP inhibitor; drug combination; synthetic lethality

DOI:  https://doi.org/10.1016/j.celrep.2022.111081
Cell Mol Life Sci. 2022 Jul 14. 79(8): 420

Molecular crowding facilitates bundling of IMPDH polymers and cytoophidium formation.

Chia-Chun Chang, Min Peng, Jiale Zhong, Ziheng Zhang, Gerson Dierley Keppeke, Li-Ying Sung, Ji-Long Liu.

  The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.

Keywords:  Cellular compartmentalization; Cytoophidium; IMPDH; Membraneless organelle; Molecular crowding

DOI:  https://doi.org/10.1007/s00018-022-04448-2
Elife. 2022 Jul 15. pii: e75741. [Epub ahead of print]11

A non-transcriptional function of Yap regulates the DNA replication program.

Rodrigo Meléndez García, Olivier Haccard, Albert Chesneau, Hemalatha Narassimprakash, Jérôme Roger, Muriel Perron, Kathrin Marheineke, Odile Bronchain.

  In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as breaks to control the DNA replication program in early embryos and post-embryonic stem cells.

Keywords:  cell biology; developmental biology; xenopus

DOI:  https://doi.org/10.7554/eLife.75741
Nature. 2022 Jul 13.

Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Yao He, He Song, Henry Chan, Baocheng Liu, Yaqiang Wang, Lukas Sušac, Z Hong Zhou, Juli Feigon.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

DOI: https://doi.org/10.1038/s41586-022-04931-7
Dev Cell. 2022 Jul 05. pii: S1534-5807(22)00448-8. [Epub ahead of print]

A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase.

Dilyana Georgieva Kirova, Kristyna Judasova, Julia Vorhauser, Thomas Zerjatke, Jacky Kieran Leung, Ingmar Glauche, Jörg Mansfeld.

  Reactive oxygen species (ROS) at the right concentration promote cell proliferation in cell culture, stem cells, and model organisms. However, the mystery of how ROS signaling is coordinated with cell cycle progression and integrated into the cell cycle control machinery on the molecular level remains unsolved. Here, we report increasing levels of mitochondrial ROS during the cell cycle in human cell lines that target cyclin-dependent kinase 2 (CDK2). Chemical and metabolic interferences with ROS production decrease T-loop phosphorylation on CDK2 and so impede its full activation and thus its efficient DNA replication. ROS regulate CDK2 activity through the oxidation of a conserved cysteine residue near the T-loop, which prevents the binding of the T-loop phosphatase KAP. Together, our data reveal how mitochondrial metabolism is coupled with DNA replication and cell cycle progression via ROS, thereby demonstrating how KAP activity toward CDKs can be cell cycle regulated.

Keywords:  CDK2; DNA replication; KAP; T-loop phosphorylation; cell cycle; cyclin-dependent kinase; metabolism; mitochondria; proliferation; reactive oxygen species

DOI:  https://doi.org/10.1016/j.devcel.2022.06.008
Biochem Biophys Res Commun. 2022 Jul 02. pii: S0006-291X(22)00966-4. [Epub ahead of print]621 137-143

Polβ modulates the expression of type I interferon via STING pathway.

Miaoling Huang, Ting Wu, Rui Liu, Meina Wang, Munan Shi, Jingyu Xin, Shan Shao, Xingqi Zhao, Ying Ma, Lili Gu, Zhigang Guo, Feiyan Pan.

  DNA Polymerase β (Polβ) is a key enzyme in base excision repair (BER), which is very important in maintaining the stability and integrity of the genome. Mutant Polβ is closely associated with carcinogenesis. However, Polβ is highly expressed in most cancers, but the underlying mechanism is not well understood. Here, we found that breast cancer cells MCF-7 with Polβ knockdown exhibited high levels of type I interferon and were easily eliminated by natural killer (NK) cells.Similarly, Polβ-mutant (R137Q) mice exhibited chronic inflammation symptoms in multiple organs and upregulated type I interferon levels. Further results showed that Polβ deficiency caused more DNA damage accumulation in cells and triggered the leakage of damaged DNA into the cytoplasm, which activated the STING/IRF3 pathway, promoted phosphorylated IRF3 translocating into the nucleus and enhanced the expression of type I interferon and proinflammatory cytokines. In addition, this effect could be eliminated by Polβ overexpression, STING inhibitor or STING knockdown. Taken together, our findings provide mechanistic insight into the role of Polβ in cancers by linking DNA repair and the inflammatory STING pathway.

Keywords:  DNA polymerase β; STING; Type I interferon; dsDNA

DOI:  https://doi.org/10.1016/j.bbrc.2022.07.005
Mol Cancer Ther. 2022 Jul 13. pii: mct.21.0891. [Epub ahead of print]

CDK7 inhibition synergizes with topoisomerase I inhibition in small cell lung cancer cells by inducing ubiquitin-mediated proteolysis of RNA polymerase II.

Yilun Sun, Yang Zhang, Christopher W Schultz, Yves Pommier, Anish Thomas.

Small-cell lung cancers (SCLCs) are highly aggressive and currently there are no available targeted therapies. To identify clinically actionable drug combinations, we analyzed our previously reported chemogenomics screens and identified a synergistically cytotoxic combination of the topoisomerase I (TOP1) inhibitor topotecan and cycle-dependent kinase 7 (CDK7) inhibitor THZ1. Topotecan causes cell death by generating TOP1-induced DNA breaks and DNA-protein crosslinks (TOP1-DPCs) that require proteolysis by the ubiquitin-proteasome pathway for their repair. We found that inhibition of the transcriptional kinase CDK7 by THZ1 induces ubiquitin-mediated proteasomal degradation of RNA polymerase II (Pol II) and prevents the proteasomal degradation of TOP1-DPCs. We also provide a mechanistic basis for combinatorial targeting of transcription using selective inhibitors of CDK7 and TOP1 in clinical trials to advance SCLC therapeutics.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0891
Cell Rep. 2022 Jul 12. pii: S2211-1247(22)00865-8. [Epub ahead of print]40(2): 111067

Resolution of R-loops by topoisomerase III-β (TOP3B) in coordination with the DEAD-box helicase DDX5.

Sourav Saha, Xi Yang, Shar-Yin Naomi Huang, Keli Agama, Simone Andrea Baechler, Yilun Sun, Hongliang Zhang, Liton Kumar Saha, Shuaikun Su, Lisa M Jenkins, Weidong Wang, Yves Pommier.

  The present study demonstrates how TOP3B is involved in resolving R-loops. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/DNA hybrid IP-western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-mass spectrometry and IP-western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with senataxin. We propose a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage.

Keywords:  CP: Molecular biology; DDX5; DNA-RNA hybrids; DNA/RNA topoisomerase 3B; R-loop; TDRD3; senataxin; topoisomerase cleavage complexes

DOI:  https://doi.org/10.1016/j.celrep.2022.111067
Bioorg Med Chem. 2022 Jul 08. pii: S0968-0896(22)00305-4. [Epub ahead of print]70 116912

Discovery of CN0 as a novel proteolysis-targeting chimera (PROTAC) degrader of PARP1 that can activate the cGAS/STING immunity pathway combined with daunorubicin.

Shanshan Lin, Guihui Tu, Zelei Yu, Qingna Jiang, Lingyu Zhang, Jingwen Liu, Quanyu Liu, Xiuwang Huang, Jianhua Xu, Youwen Lin, Yang Liu, Lixian Wu.

  Poly ADP-ribose polymerase 1 (PARP1) plays an essential role in DNA repair signaling, rendering it an attractive target for cancer treatment. Despite the success of PARP1 inhibitors (PARPis), only a few patients can currently benefit from PARPis. Moreover, drug resistance to PARPis occurs during clinical treatment. Natural and acquired resistance to PARPis has forced us to seek new therapeutic approaches that target PARP1. Here, we synthesized a series of compounds by proteolysis-targeting chimera (PROTAC) technology to directly degrade the PARP1 protein. We found that CN0 (compound 3) with no polyethylene glycol (PEG) linker can degrade the PARP1 protein through the proteasome pathway. More importantly, CN0 could inhibit DNA damage repair, resulting in highly efficient accumulation of cytosolic DNA fragments due to unresolved unrepaired DNA lesions when combined with daunorubicin (DNR). Therefore, CN0 can activate the cyclic GMP-AMP synthase/stimulator of the interferon gene (cGAS/STING) pathway of innate immunity and then spread the resulting inflammatory signals, thereby reshaping the tumor microenvironment, which may eventually enhance T cell killing of tumor cells.

Keywords:  DNA repair; Daunorubicin; PARP1; PROTAC; STING

DOI:  https://doi.org/10.1016/j.bmc.2022.116912
Cancer Gene Ther. 2022 Jul 15.

MicroRNA-21 guide and passenger strand regulation of adenylosuccinate lyase-mediated purine metabolism promotes transition to an EGFR-TKI-tolerant persister state.

Wen Cai Zhang, Nicholas Skiados, Fareesa Aftab, Cerena Moreno, Luis Silva, Paul Joshua Anthony Corbilla, John M Asara, Aaron N Hata, Frank J Slack.

In EGFR-mutant lung cancer, drug-tolerant persister cells (DTPCs) show prolonged survival when receiving EGFR tyrosine kinase inhibitor (TKI) treatments. They are a likely source of drug resistance, but little is known about how these cells tolerate drugs. Ribonucleic acids (RNAs) molecules control cell growth and stress responses. Nucleic acid metabolism provides metabolites, such as purines, supporting RNA synthesis and downstream functions. Recently, noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), have received attention due to their capacity to repress gene expression via inhibitory binding to downstream messenger RNAs (mRNAs). Here, our study links miRNA expression to purine metabolism and drug tolerance. MiR-21-5p (guide strand) is a commonly upregulated miRNA in disease states, including cancer and drug resistance. However, the expression and function of miR-21-3p (passenger strand) are not well understood. We found that upregulation of miR-21-5p and miR-21-3p tune purine metabolism leading to increased drug tolerance. Metabolomics data demonstrated that purine metabolism was the top pathway in the DTPCs compared with the parental cells. The changes in purine metabolites in the DTPCs were partially rescued by targeting miR-21. Analysis of protein levels in the DTPCs showed that reduced expression of adenylosuccinate lyase (ADSL) was reversed after the miR-21 knockdown. ADSL is an essential enzyme in the de novo purine biosynthesis pathway by converting succino-5-aminoimidazole-4-carboxamide riboside (succino-AICAR or SAICAR) to AICAR (or acadesine) as well as adenylosuccinate to adenosine monophosphate (AMP). In the DTPCs, miR-21-5p and miR-21-3p repress ADSL expression. The levels of top decreased metabolite in the DTPCs, AICAR was reversed when miR-21 was blocked. AICAR induced oxidative stress, evidenced by increased reactive oxygen species (ROS) and reduced expression of nuclear factor erythroid-2-related factor 2 (NRF2). Concurrently, miR-21 knockdown induced ROS generation. Therapeutically, a combination of AICAR and osimertinib increased ROS levels and decreased osimertinib-induced NRF2 expression. In a MIR21 knockout mouse model, MIR21 loss-of-function led to increased purine metabolites but reduced ROS scavenging capacity in lung tissues in physiological conditions. Our data has established a link between ncRNAs, purine metabolism, and the redox imbalance pathway. This discovery will increase knowledge of the complexity of the regulatory RNA network and potentially enable novel therapeutic options for drug-resistant patients.

DOI: https://doi.org/10.1038/s41417-022-00504-y
Cancer Gene Ther. 2022 Jul 15.

Nrf2 overexpression increases the resistance of acute myeloid leukemia to cytarabine by inhibiting replication factor C4.

Tianzhen Hu, Chengyun Pan, Tianzhuo Zhang, Ming Ni, Weili Wang, Siyu Zhang, Ying Chen, Jishi Wang, Qin Fang.

Drug resistance is a key factor in the treatment failure of acute myeloid leukemia (AML). Nuclear factor E2-related factor 2 (Nrf2) plays a crucial role in tumor chemotherapy resistance. However, the potential mechanism of Nrf2 regulating DNA mismatch repair (MMR) pathway to mediate gene-instability drug resistance in AML is still unclear. Here, it was found that Nrf2 expression was closely related to the disease progression of AML as well as highly expressed in AML patients with poor prognostic gene mutations. Meanwhile, it was also found that the expression of Nrf2 was significantly negatively correlated with DNA MMR gene replication factor C4 (RFC4) in AML. CHIP analysis combined with luciferase reporter gene results further showed that Nrf2 may inhibit the expression of RFC4 by its interaction with the RFC4 promoter. In vitro and vivo experiments showed that the overexpression of Nrf2 decreased the killing effect of chemotherapy drug cytarabine (Ara-C) on leukemia cells and inhibited the expression of RFC4. Mechanistically, The result that Nrf2-RFC4 axis mediated AML genetic instability drug resistance might be received by activating the JNK/NF-κB signaling pathway. Taken together, these findings may provide a new idea for improving AML drug resistance.

DOI: https://doi.org/10.1038/s41417-022-00501-1
Nat Microbiol. 2022 Jul 11.

Bacteria deplete deoxynucleotides to defend against bacteriophage infection.

Nitzan Tal, Adi Millman, Avigail Stokar-Avihail, Taya Fedorenko, Azita Leavitt, Sarah Melamed, Erez Yirmiya, Carmel Avraham, Alexander Brandis, Tevie Mehlman, Gil Amitai, Rotem Sorek.

DNA viruses and retroviruses consume large quantities of deoxynucleotides (dNTPs) when replicating. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral lifecycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. Here, we report that bacteria use a similar strategy to defend against bacteriophage infection. We identify a family of defensive bacterial deoxycytidine triphosphate (dCTP) deaminase proteins that convert dCTP into deoxyuracil nucleotides in response to phage infection. We also identify a family of phage resistance genes that encode deoxyguanosine triphosphatase (dGTPase) enzymes, which degrade dGTP into phosphate-free deoxyguanosine and are distant homologues of human SAMHD1. Our results suggest that bacterial defensive proteins deplete specific deoxynucleotides (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Our study shows that manipulation of the dNTP pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

DOI: https://doi.org/10.1038/s41564-022-01158-0