bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2022–07–10
23 papers selected by
Sean Rudd, Karolinska Institutet
  1. Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation and ssDNA binding kinetics.
  2. Mechanisms and Regulation of DNA-Protein Crosslink Repair During DNA Replication by SPRTN Protease.
  3. Revisiting the Function of p21CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability.
  4. Context-dependent pro- and anti-resection roles of ZKSCAN3 in the regulation of fork processing during replication stress.
  5. Structures of LIG1 that engage with mutagenic mismatches inserted by polβ in base excision repair.
  6. Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex.
  7. Increased Resection at DSBs in G2-Phase Is a Unique Phenotype Associated with DNA-PKcs Defects That Is Not Shared by Other Factors of c-NHEJ.
  8. Targeting the DNA damage response beyond poly(ADP-ribose) polymerase inhibitors: novel agents and rational combinations.
  9. A synthetic lethal strategy using PARP and ATM inhibition for overcoming trastuzumab resistance in HER2-positive cancers.
  10. Global metabolic alterations in colorectal cancer cells during irinotecan-induced DNA replication stress.
  11. The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase.
  12. Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective.
  13. Molecular Link between DNA Damage Response and Microtubule Dynamics.
  14. DNA damage and metabolic mechanisms of cancer drug resistance.
  15. Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome.
  16. Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.
  17. Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation.
  18. Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.
  19. Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis.
  20. Captured snapshots of PARP1 in the active state reveal the mechanics of PARP1 allostery.
  21. RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells.
  22. Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype.
  23. Stabilization of SAMHD1 by NONO is crucial for Ara-C resistance in AML.
  1. Nucleic Acids Res. 2022 Jul 08. pii: gkac573. [Epub ahead of print]
    Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation and ssDNA binding kinetics.
    Benjamin Orris, Kevin W Huynh, Mark Ammirati, Seungil Han, Ben Bolaños, Jason Carmody, Matthew D Petroski, Benedikt Bosbach, David J Shields, James T Stivers.
      SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart. Although phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (∼15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial dynamics in the C-terminal domain (which contains T592), which could be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.
    DOI:  https://doi.org/10.1093/nar/gkac573
  2. Front Mol Biosci. 2022 ;9 916697
    Mechanisms and Regulation of DNA-Protein Crosslink Repair During DNA Replication by SPRTN Protease.
    Megan Perry, Gargi Ghosal.
      DNA-protein crosslinks (DPCs) are deleterious DNA lesions that occur when proteins are covalently crosslinked to the DNA by the action of variety of agents like reactive oxygen species, aldehydes and metabolites, radiation, and chemotherapeutic drugs. Unrepaired DPCs are blockades to all DNA metabolic processes. Specifically, during DNA replication, replication forks stall at DPCs and are vulnerable to fork collapse, causing DNA breakage leading to genome instability and cancer. Replication-coupled DPC repair involves DPC degradation by proteases such as SPRTN or the proteasome and the subsequent removal of DNA-peptide adducts by nucleases and canonical DNA repair pathways. SPRTN is a DNA-dependent metalloprotease that cleaves DPC substrates in a sequence-independent manner and is also required for translesion DNA synthesis following DPC degradation. Biallelic mutations in SPRTN cause Ruijs-Aalfs (RJALS) syndrome, characterized by hepatocellular carcinoma and segmental progeria, indicating the critical role for SPRTN and DPC repair pathway in genome maintenance. In this review, we will discuss the mechanism of replication-coupled DPC repair, regulation of SPRTN function and its implications in human disease and cancer.
    Keywords:  DPC; DPC proteolysis; Ruijs-Aalfs syndrome; SPRTN protease; translesion synthesis (TLS)
    DOI:  https://doi.org/10.3389/fmolb.2022.916697
  3. Int J Mol Sci. 2022 Jun 24. pii: 7058. [Epub ahead of print]23(13):
    Revisiting the Function of p21CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability.
    Giulio Ticli, Ornella Cazzalini, Lucia A Stivala, Ennio Prosperi.
      The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms.
    Keywords:  DNA damage response; DNA repair; PCNA; p21CDKN1A; protein degradation
    DOI:  https://doi.org/10.3390/ijms23137058
  4. J Biol Chem. 2022 Jun 29. pii: S0021-9258(22)00657-3. [Epub ahead of print] 102215
    Context-dependent pro- and anti-resection roles of ZKSCAN3 in the regulation of fork processing during replication stress.
    Zheng Yang, Delphine Sangotokun Lemacon, Shan Li, Abigael Cheruiyot, Lingzhen Kong, Ke Tan, Chen Cheng, Ecenur Turkay, Dalin He, Zhongsheng You.
      Uncontrolled resection of replication forks under stress can cause genomic instability and influence cancer formation. Extensive fork resection has also been implicated in the chemosensitivity of "BReast CAncer gene" BRCA-deficient cancers. However, how fork resection is controlled in different genetic contexts and how it affects chromosomal stability and cell survival remains incompletely understood. Here, we report a novel function of the transcription repressor ZKSCAN3 in fork protection and chromosomal stability maintenance under replication stress. We show disruption of ZKSCAN3 function causes excessive resection of replication forks by the exonuclease Exo1 and homologous DNA recombination/repair protein Mre11 following fork reversal. Interestingly, in BRCA1-deficient cells, we found ZKSCAN3 actually promotes fork resection upon replication stress. We demonstrate these anti- and pro-resection roles of ZKSCAN3, consisting of a SCAN box, Kruppel-associated box (KRAB), and zinc finger (ZNF) domain, are mediated by its SCAN box domain and do not require the KRAB or ZNF domains, suggesting that the transcriptional function of ZKSCAN3 is not involved. Furthermore, despite the severe impact on fork structure and chromosomal stability, depletion of ZKSCAN3 did not affect the short-term survival of BRCA1-proficient or -deficient cells after treatment with cancer drugs hydroxyurea, PARPi, or cisplatin. Our findings reveal a unique relationship between ZKSCAN3 and BRCA1 in fork protection and add to our understanding of the relationships between replication fork protection, chromosomal instability, and chemosensitivity.
    Keywords:  BRCA1; Replication stress; ZKSCAN3; chemosensitivity; fork resection
    DOI:  https://doi.org/10.1016/j.jbc.2022.102215
  5. Nat Commun. 2022 Jul 05. 13(1): 3860
    Structures of LIG1 that engage with mutagenic mismatches inserted by polβ in base excision repair.
    Qun Tang, Mitchell Gulkis, Robert McKenna, Melike Çağlayan.
      DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) β during downstream steps of the base excision repair (BER) pathway. However, how LIG1 discriminates against the mutagenic 3'-mismatches incorporated by polβ at atomic resolution remains undefined. Here, we determine the X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncover the ligase strategies that favor or deter the ligation of base substitution errors. Our structures reveal that the LIG1 active site can accommodate a G:T mismatch in the wobble conformation, where an adenylate (AMP) is transferred to the 5'-phosphate of a nick (DNA-AMP), while it stays in the LIG1-AMP intermediate during the initial step of the ligation reaction in the presence of an A:C mismatch at the 3'-strand. Moreover, we show mutagenic ligation and aberrant nick sealing of dG:T and dA:C mismatches, respectively. Finally, we demonstrate that AP-endonuclease 1 (APE1), as a compensatory proofreading enzyme, removes the mismatched bases and interacts with LIG1 at the final BER steps. Our overall findings provide the features of accurate versus mutagenic outcomes coordinated by a multiprotein complex including polβ, LIG1, and APE1 to maintain efficient repair.
    DOI:  https://doi.org/10.1038/s41467-022-31585-w
  6. Nucleic Acids Res. 2022 Jul 08. pii: gkac583. [Epub ahead of print]
    Strand annealing and motor driven activities of SMARCAL1 and ZRANB3 are stimulated by RAD51 and the paralog complex.
    Swagata Halder, Lepakshi Ranjha, Angelo Taglialatela, Alberto Ciccia, Petr Cejka.
      SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.
    DOI:  https://doi.org/10.1093/nar/gkac583
  7. Cells. 2022 Jul 02. pii: 2099. [Epub ahead of print]11(13):
    Increased Resection at DSBs in G2-Phase Is a Unique Phenotype Associated with DNA-PKcs Defects That Is Not Shared by Other Factors of c-NHEJ.
    Huaping Xiao, Fanghua Li, Emil Mladenov, Aashish Soni, Veronika Mladenova, Bing Pan, Rositsa Dueva, Martin Stuschke, Beate Timmermann, George Iliakis.
      The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 Gy. Increased resection and HR engagement with decreasing DSB-load generate a conundrum in a classical non-homologous end-joining (c-NHEJ)-dominated cell and suggest a mechanism adaptively facilitating resection. We report that ablation of DNA-PKcs causes hyper-resection, implicating DNA-PK in the underpinning mechanism. However, hyper-resection in DNA-PKcs-deficient cells can also be an indirect consequence of their c-NHEJ defect. Here, we report that all tested DNA-PKcs mutants show hyper-resection, while mutants with defects in all other factors of c-NHEJ fail to do so. This result rules out the model of c-NHEJ versus HR competition and the passive shift from c-NHEJ to HR as the causes of the increased resection and suggests the integration of DNA-PKcs into resection regulation. We develop a model, compatible with the results of others, which integrates DNA-PKcs into resection regulation and HR for a subset of DSBs. For these DSBs, we propose that the kinase remains at the break site, rather than the commonly assumed autophosphorylation-mediated removal from DNA ends.
    Keywords:  DNA end-resection; DNA-PKcs; DSB repair; c-NHEJ; ionizing radiation
    DOI:  https://doi.org/10.3390/cells11132099
  8. Curr Opin Oncol. 2022 Jul 06.
    Targeting the DNA damage response beyond poly(ADP-ribose) polymerase inhibitors: novel agents and rational combinations.
    Natalie Y L Ngoi, Shannon N Westin, Timothy A Yap.
       PURPOSE OF REVIEW: Poly(ADP-ribose) polymerase (PARP) inhibitors have transformed treatment paradigms in multiple cancer types defined by homologous recombination deficiency (HRD) and have become the archetypal example of synthetic lethal targeting within the DNA damage response (DDR). Despite this success, primary and acquired resistance to PARP inhibition inevitability threaten the efficacy and durability of response to these drugs. Beyond PARP inhibitors, recent advances in large-scale functional genomic screens have led to the identification of a steadily growing list of genetic dependencies across the DDR landscape. This has led to a wide array of novel synthetic lethal targets and corresponding inhibitors, which hold promise to widen the application of DDR inhibitors beyond HRD and potentially address PARP inhibitor resistance.
    RECENT FINDINGS: In this review, we describe key synthetic lethal interactions that have been identified across the DDR landscape, summarize the early phase clinical development of the most promising DDR inhibitors, and highlight relevant combinations of DDR inhibitors with chemotherapy and other novel cancer therapies, which are anticipated to make an impact in rationally selected patient populations.
    SUMMARY: The DDR landscape holds multiple opportunities for synthetic lethal targeting with multiple novel DDR inhibitors being evaluated on early phase clinical trials. Key challenges remain in optimizing the therapeutic window of ATR and WEE1 inhibitors as monotherapy and in combination approaches.
    DOI:  https://doi.org/10.1097/CCO.0000000000000867
  9. Oncogene. 2022 Jul 07.
    A synthetic lethal strategy using PARP and ATM inhibition for overcoming trastuzumab resistance in HER2-positive cancers.
    Kyoung-Seok Oh, Ah-Rong Nam, Ju-Hee Bang, Hye-Rim Seo, Jae-Min Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh.
      Despite its clinical efficacy in HER2-positive cancers, resistance to trastuzumab inevitably occurs. The DNA damage response (DDR) pathway is essential for maintaining genomic stability and cell survival. However, the role of the DDR pathway in HER2-positive tumors and trastuzumab resistance remains elusive. In this study, we verified that increased PARP1 expression in trastuzumab-resistant (TR) cells, owing to its augmented stability by escape from proteasomal degradation, confers tolerability to trastuzumab-induced DNA damage. Interruption of PARP1 in TR cells restrains its cellular growth, while simultaneously activating ATM to retain its genome stability. Dual inhibition of PARP and ATM induces synthetic lethality in TR cells by favoring the toxic NHEJ pathway instead of the HRR pathway. Our results highlight the potential of clinical development of DDR-targeting strategies for trastuzumab-resistant HER2-positive cancer patients.
    DOI:  https://doi.org/10.1038/s41388-022-02384-w
  10. Cancer Metab. 2022 Jul 04. 10(1): 10
    Global metabolic alterations in colorectal cancer cells during irinotecan-induced DNA replication stress.
    Christian Marx, Jürgen Sonnemann, Oliver D K Maddocks, Lisa Marx-Blümel, Mandy Beyer, Doerte Hoelzer, René Thierbach, Claudia Maletzki, Michael Linnebacher, Thorsten Heinzel, Oliver H Krämer.
       BACKGROUND: Metabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently.
    METHODS: We incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites.
    RESULTS: Chemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions.
    CONCLUSION: Drugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells. The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.
    Keywords:  Adaptation; Colorectal cancer; Glucose; Irinotecan; Metabolism; Warburg effect; p53
    DOI:  https://doi.org/10.1186/s40170-022-00286-9
  11. J Biol Chem. 2022 Jul 04. pii: S0021-9258(22)00676-7. [Epub ahead of print] 102234
    The p97 segregase cofactor Ubxn7 facilitates replisome disassembly during S-phase.
    Zeynep Tarcan, Divyasree Poovathumkadavil, Aggeliki Skagia, Agnieszka Gambus.
      Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. While we are beginning to understand the molecular mechanism for assembly of the eukaryotic DNA replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Recently, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading, and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases have now been shown to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here, using Xenopus laevis egg extract cell-free system and biochemical approaches, we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly during S-phase. We show only Ubxn7, however, facilitates efficient replisome disassembly. Ubxn7 delivers this role through its interaction via independent domains with both Cul2Lrr1 and p97 to allow coupling between Mcm7 ubiquitylation and its removal from chromatin. Our data therefore characterize Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates and a rationale for the efficacy of the Cul2Lrr1 replisome unloading pathway in unperturbed S-phase.
    Keywords:  DNA replication; Termination of DNA replication; Xenopus laevis; p97 segregase; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102234
  12. Cancers (Basel). 2022 Jun 25. pii: 3113. [Epub ahead of print]14(13):
    Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective.
    Syed Saqib Ali, Ruchika Raj, Tejinder Kaur, Brenna Weadick, Debasis Nayak, Minnsung No, Jane Protos, Hannah Odom, Kajal Desai, Avinash K Persaud, Joanne Wang, Rajgopal Govindarajan.
      Anticancer nucleoside analogs produce adverse, and at times, dose-limiting hematological toxicities that can compromise treatment efficacy, yet the mechanisms of such toxicities are poorly understood. Recently, cellular nucleoside transport has been implicated in normal blood cell formation with studies from nucleoside transporter-deficient mice providing additional insights into the regulation of mammalian hematopoiesis. Furthermore, several idiopathic human genetic disorders have revealed nucleoside transport as an important component of mammalian hematopoiesis because mutations in individual nucleoside transporter genes are linked to various hematological abnormalities, including anemia. Here, we review recent developments in nucleoside transporters, including their transport characteristics, their role in the regulation of hematopoiesis, and their potential involvement in the occurrence of adverse hematological side effects due to nucleoside drug treatment. Furthermore, we discuss the putative mechanisms by which aberrant nucleoside transport may contribute to hematological abnormalities and identify the knowledge gaps where future research may positively impact treatment outcomes for patients undergoing various nucleoside analog therapies.
    Keywords:  anemia; drug; hematological; myelosuppression; nucleoside; toxicity; transporter
    DOI:  https://doi.org/10.3390/cancers14133113
  13. Int J Mol Sci. 2022 Jun 23. pii: 6986. [Epub ahead of print]23(13):
    Molecular Link between DNA Damage Response and Microtubule Dynamics.
    Jung Min Kim.
      Microtubules are major components of the cytoskeleton that play important roles in cellular processes such as intracellular transport and cell division. In recent years, it has become evident that microtubule networks play a role in genome maintenance during interphase. In this review, we highlight recent advances in understanding the role of microtubule dynamics in DNA damage response and repair. We first describe how DNA damage checkpoints regulate microtubule organization and stability. We then highlight how microtubule networks are involved in the nuclear remodeling following DNA damage, which leads to changes in chromosome organization. Lastly, we discuss how microtubule dynamics participate in the mobility of damaged DNA and promote consequent DNA repair. Together, the literature indicates the importance of microtubule dynamics in genome organization and stability during interphase.
    Keywords:  DNA damage response; DNA repair; centrosome; chromatin mobility; microtubules; nuclear reorganization
    DOI:  https://doi.org/10.3390/ijms23136986
  14. Cancer Drug Resist. 2022 ;5(2): 368-379
    DNA damage and metabolic mechanisms of cancer drug resistance.
    Deanna Tiek, Shi-Yuan Cheng.
      Cancer drug resistance is one of the main barriers to overcome to ensure durable treatment responses. While many pivotal advances have been made in first combination therapies, then targeted therapies, and now broadening out to immunomodulatory drugs or metabolic targeting compounds, drug resistance is still ultimately universally fatal. In this brief review, we will discuss different strategies that have been used to fight drug resistance from synthetic lethality to tumor microenvironment modulation, focusing on the DNA damage response and tumor metabolism both within tumor cells and their surrounding microenvironment. In this way, with a better understanding of both targetable mutations in combination with the metabolism, smarter drugs may be designed to combat cancer drug resistance.
    Keywords:  Cancer drug resistance; DNA damage; DNA repair; drug resistance; hypoxia; metabolism; overcoming resistance; synthetic lethality
    DOI:  https://doi.org/10.20517/cdr.2021.148
  15. Nat Cell Biol. 2022 Jul 04.
    Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome.
    Yingying Meng, Guliang Wang, Hongjuan He, Kin H Lau, Allison Hurt, Brianna J Bixler, Andrea Parham, Seung-Gi Jin, Xingzhi Xu, Karen M Vasquez, Gerd P Pfeifer, Piroska E Szabó.
      Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.
    DOI:  https://doi.org/10.1038/s41556-022-00941-9
  16. Front Cell Dev Biol. 2022 ;10 843121
    Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.
    Anna K Miller, Guogen Mao, Breanna G Knicely, Hannah G Daniels, Christine Rahal, Christopher D Putnam, Richard D Kolodner, Eva M Goellner.
      DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.
    Keywords:  HLTF; SHPRH; alkylating agent MNNG; binding motif; mismatch repair (MMR); rad5
    DOI:  https://doi.org/10.3389/fcell.2022.843121
  17. Nucleic Acids Res. 2022 Jul 08. pii: gkac555. [Epub ahead of print]
    Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation.
    Guillaume Guilbaud, Pierre Murat, Helen S Wilkes, Leticia Koch Lerner, Julian E Sale, Torsten Krude.
      Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered 'heavy-light' (HL) by incorporation of BrdUTP while unreplicated DNA remains 'light-light' (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.
    DOI:  https://doi.org/10.1093/nar/gkac555
  18. Cell Rep Methods. 2022 Jun 20. 2(6): 100237
    Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.
    Pin-Rui Su, Li You, Cecile Beerens, Karel Bezstarosti, Jeroen Demmers, Martin Pabst, Roland Kanaar, Cheng-Chih Hsu, Miao-Ping Chien.
      Single-cell proteomics has the potential to decipher tumor heterogeneity, and a method like single-cell proteomics by mass spectrometry (SCoPE-MS) allows profiling several tens of single cells for >1,000 proteins per cell. This method, however, cannot link the proteome of individual cells with phenotypes of interest. Here, we developed a microscopy-based functional single-cell proteomic-profiling technology, called FUNpro, to address this. FUNpro enables screening, identification, and isolation of single cells of interest in a real-time fashion, even if the phenotypes are dynamic or the cells of interest are rare. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified the PDS5A protein contributing to the abnormal DDR dynamics and helping the cells survive after IR.
    Keywords:  53BP1; DDR foci dynamics; DNA damage response; PDS5A; functional single-cell selection; phenotype-to-proteome linking; phototagging; single-cell proteomics; tumor heterogeneity; ultrawide field-of-view optical microscope
    DOI:  https://doi.org/10.1016/j.crmeth.2022.100237
  19. Sci Rep. 2022 Jul 05. 12(1): 11344
    Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis.
    Barbara Tomic, Tomislav Smoljo, Hrvoje Lalic, Vilma Dembitz, Josip Batinic, Drago Batinic, Antonio Bedalov, Dora Visnjic.
      Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.
    DOI:  https://doi.org/10.1038/s41598-022-15520-z
  20. Mol Cell. 2022 Jun 28. pii: S1097-2765(22)00570-6. [Epub ahead of print]
    Captured snapshots of PARP1 in the active state reveal the mechanics of PARP1 allostery.
    Élise Rouleau-Turcotte, Dragomir B Krastev, Stephen J Pettitt, Christopher J Lord, John M Pascal.
      PARP1 rapidly detects DNA strand break damage and allosterically signals break detection to the PARP1 catalytic domain to activate poly(ADP-ribose) production from NAD+. PARP1 activation is characterized by dynamic changes in the structure of a regulatory helical domain (HD); yet, there are limited insights into the specific contributions that the HD makes to PARP1 allostery. Here, we have determined crystal structures of PARP1 in isolated active states that display specific HD conformations. These captured snapshots and biochemical analysis illustrate HD contributions to PARP1 multi-domain and high-affinity interaction with DNA damage, provide novel insights into the mechanics of PARP1 allostery, and indicate how HD active conformations correspond to alterations in the catalytic region that reveal the active site to NAD+. Our work deepens the understanding of PARP1 catalytic activation, the dynamics of the binding site of PARP inhibitor compounds, and the mechanisms regulating PARP1 retention on DNA damage.
    Keywords:  ADP-ribose; DNA strand breaks; PARP enzymes; PARP inhibitors; PARP1; allostery; poly(ADP-ribose)
    DOI:  https://doi.org/10.1016/j.molcel.2022.06.011
  21. Cancer Drug Resist. 2022 ;5(2): 401-414
    RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells.
    Pascale Palassin, Marion Lapierre, Sandrine Bonnet, Marie-Jeanne Pillaire, Balázs Győrffy, Catherine Teyssier, Stéphan Jalaguier, Jean-Sébastien Hoffmann, Vincent Cavaillès, Audrey Castet-Nicolas.
      Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites. Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined. Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC. Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity.
    Keywords:  Colorectal cancer; Pol Kappa; RIP140; genome stability; translesion DNA synthesis polymerase
    DOI:  https://doi.org/10.20517/cdr.2021.133
  22. J Vis Exp. 2022 Jun 17.
    Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype.
    Saswati Das, Raveen Stephen Stallon Illangeswaran, Smitha Ijee, Sanjay Kumar, Shaji Ramachandran Velayudhan, Poonkuzhali Balasubramanian.
      Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.
    DOI:  https://doi.org/10.3791/63383
  23. Cell Death Dis. 2022 Jul 08. 13(7): 590
    Stabilization of SAMHD1 by NONO is crucial for Ara-C resistance in AML.
    Feifei Zhang, Jun Sun, Xiaofeng Tang, Yiping Liang, Quanhui Jiao, Bo Yu, Zhengzai Dai, Xuhui Yuan, Jiayu Li, Jinhua Yan, Zhiping Zhang, Song Fan, Min Wang, Haiyan Hu, Changhua Zhang, Xiao-Bin Lv.
      Cytarabine (Ara-C) is the first-line drug for the treatment of acute myelogenous leukemia (AML). However, resistance eventually develops, decreasing the efficacy of Ara-C in AML patients. The expression of SAMHD1, a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, has been reported to be elevated in Ara-C-resistant AML patients and to play a crucial role in mediating Ara-C resistance in AML. However, the mechanism by which SAMHD1 is upregulated in resistant AML remains unknown. In this study, NONO interacted with and stabilized SAMHD1 by inhibiting DCAF1-mediated ubiquitination/degradation of SAMHD1. Overexpression of NONO increased SAMHD1 expression and reduced the sensitivity of AML cells to Ara-C, and downregulation of NONO had the opposite effects. In addition, the DNA-damaging agents DDP and adriamycin (ADM) reduced NONO/SAMHD1 expression and sensitized AML cells to Ara-C. More importantly, NONO was upregulated in Ara-C-resistant AML cells, resulting in increased SAMHD1 expression in resistant AML cells, and DDP and ADM treatment resensitized resistant AML cells to Ara-C. This study revealed the mechanism by which SAMHD1 is upregulated in Ara-C-resistant AML cells and provided novel therapeutic strategies for Ara-C-resistant AML.
    DOI:  https://doi.org/10.1038/s41419-022-05023-0
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