bims-numges Biomed News
on Nucleotide metabolism and genome stability
Issue of 2021‒08‒08
forty-six papers selected by
Sean Rudd
Karolinska Institutet
  1. Targeting leukemia-specific dependence on the de novo purine synthesis pathway.
  2. Innate immune response orchestrates phosphoribosyl pyrophosphate synthetases to support DNA repair.
  3. Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy.
  4. Epithelial Cell Transforming Factor ECT2 Is an Important Regulator of DNA Double-Strand Break Repair and Genome Stability.
  5. Advances in SUMO-based regulation of homologous recombination.
  6. The DNA-binding protein CST associates with the cohesin complex and promotes chromosome cohesion.
  7. Functional Roles of Homologous Recombination and Non-Homologous End Joining in DNA Damage Response and Microevolution in Cryptococcus neoformans.
  8. ASTE1 promotes shieldin-complex-mediated DNA repair by attenuating end resection.
  9. GRB2 enforces homology-directed repair initiation by MRE11.
  10. Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway.
  11. The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair.
  12. Serine ADP-ribosylation in DNA-damage response regulation.
  13. Exploiting DNA repair pathways for tumor sensitization, mitigation of resistance, and normal tissue protection in radiotherapy.
  14. Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events.
  15. Nonhomologous DNA end joining of nucleosomal substrates in a purified system.
  16. Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies.
  17. Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells.
  18. Inhibition of WEE1 Potentiates Sensitivity to PARP Inhibitor in Biliary Tract Cancer.
  19. Inhibiting homologous recombination by targeting RAD51 protein.
  20. Dpb4 promotes resection of DNA double-strand breaks and checkpoint activation by acting in two different protein complexes.
  21. Dealing with transcription-blocking DNA damage: Repair mechanisms, RNA polymerase II processing and human disorders.
  22. Targeting DNA Damage Response and Repair to Enhance Therapeutic Index in Cisplatin-Based Cancer Treatment.
  23. Understanding BRCA2 Function as a Tumor Suppressor Based on Domain-Specific Activities in DNA Damage Responses.
  24. Non-canonical function of nuclear PTEN and its implication on tumorigenesis.
  25. Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.
  26. Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.
  27. High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.
  28. Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.
  29. DNA Polymerase Iota Promotes Esophageal Squamous Cell Carcinoma Proliferation Through Erk-OGT-Induced G6PD Overactivation.
  30. BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.
  31. The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.
  32. Wild-type IDH2 protects nuclear DNA from oxidative damage and is a potential therapeutic target in colorectal cancer.
  33. Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.
  34. Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.
  35. Hydroxyurea-The Good, the Bad and the Ugly.
  36. Molecular targeted drugs resistance impairs double-strand break repair and sensitizes ER-positive breast cancer to PARP inhibitors.
  37. Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.
  38. Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies.
  39. BRCA2 Promotes Spontaneous Homologous Recombination In Vivo.
  40. ASTE1 cutting to block DNA end resection.
  41. Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.
  42. Dual PARP and RAD51 Inhibitory Drug Conjugates Show Synergistic and Selective Effects on Breast Cancer Cells.
  43. Targeting Replication Stress Using CHK1 Inhibitor Promotes Innate and NKT Cell Immune Responses and Tumour Regression.
  44. SMC complexes are guarded by the SUMO protease Ulp2 against SUMO-chain-mediated turnover.
  45. Genetic insights into biological mechanisms governing human ovarian ageing.
  46. A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.
  1. Leukemia. 2021 Aug 03.
    Targeting leukemia-specific dependence on the de novo purine synthesis pathway.
    Takuji Yamauchi, Kohta Miyawaki, Yuichiro Semba, Masatomo Takahashi, Yoshihiro Izumi, Jumpei Nogami, Fumihiko Nakao, Takeshi Sugio, Kensuke Sasaki, Luca Pinello, Daniel E Bauer, Takeshi Bamba, Koichi Akashi, Takahiro Maeda.
      Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival.
    DOI:  https://doi.org/10.1038/s41375-021-01369-0
  2. Cell Metab. 2021 Jul 28. pii: S1550-4131(21)00325-9. [Epub ahead of print]
    Innate immune response orchestrates phosphoribosyl pyrophosphate synthetases to support DNA repair.
    Rui Liu, Jingyi Li, Jichun Shao, Jong-Ho Lee, Xuemei Qiu, Yanxuan Xiao, Bowen Zhang, Yilong Hao, Mi Li, Qianming Chen.
      Ionizing radiation-induced DNA damages cause genome instability and are highly cytotoxic. Deoxyribonucleotide metabolism provides building blocks for DNA repair. Nevertheless, how deoxyribonucleotide metabolism is timely regulated to coordinate with DNA repair remains elusive. Here, we show that ionizing radiation results in TBK1-mediated phosphorylation of phosphoribosyl pyrophosphate synthetase (PRPS)1/2 at T228, thereby enhancing PRPS1/2 catalytic activity and promoting deoxyribonucleotide synthesis. DNA damage-elicited activation of cGAS/STING axis and ATM-mediated PRPS1/2 S16 phosphorylation are required for PRPS1/2 T228 phosphorylation under ionizing radiation. Furthermore, T228 phosphorylation overrides allosteric regulator-mediated effects and preserves PRPS1/2 with high activity. The expression of non-phosphorylatable PRPS1/2 mutants or inhibition of cGAS/STING axis counteracts ionizing radiation-induced PRPS1/2 activation, deoxyribonucleotide synthesis, and DNA repair, and further impairs cell viability. This study highlights a novel and important mechanism underlying an innate immune response-guided deoxyribonucleotide metabolism, which supports DNA repair.
    Keywords:  ATM; DNA repair; STING; TBK1; cGAS; innate immune; ionizing radiation; nucleotide metabolism; phosphoribosyl pyrophosphate synthetase
    DOI:  https://doi.org/10.1016/j.cmet.2021.07.009
  3. Leukemia. 2021 Aug 02.
    Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy.
    Yana Pikman, Nicole Ocasio-Martinez, Gabriela Alexe, Boris Dimitrov, Samuel Kitara, Frances F Diehl, Amanda L Robichaud, Amy Saur Conway, Linda Ross, Angela Su, Frank Ling, Jun Qi, Giovanni Roti, Caroline A Lewis, Alexandre Puissant, Matthew G Vander Heiden, Kimberly Stegmaier.
      Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.
    DOI:  https://doi.org/10.1038/s41375-021-01361-8
  4. J Biol Chem. 2021 Jul 31. pii: S0021-9258(21)00838-3. [Epub ahead of print] 101036
    Epithelial Cell Transforming Factor ECT2 Is an Important Regulator of DNA Double-Strand Break Repair and Genome Stability.
    Cheng Cao, Peiyi Han, Ling Liu, Yiman Tang, Shanshan Tian, Kai Zhang, Lei Shi, Zhiqiang Liu, Dexiang Zhuo, Wenshu Ge, Wenchen Gong.
      Proteins containing BRCA1 C-terminal (BRCT) domains play crucial roles in response to and repair of DNA damage. Epithelial cell transforming factor ECT2 is a member of the BRCT protein family, but it is not known if ECT2 directly contributes to DNA repair. In this study, we report that ECT2 is recruited to DNA lesions in a poly(ADP ribose) polymerase 1 (PARP1)-dependent manner. We showed that ECT2 physically associates with KU70-KU80 and BRCA1, proteins involved in nonhomologous end-joining and homologous recombination, respectively. ECT2 deficiency impairs the recruitment of KU70 and BRCA1 to DNA damage sites, resulting in defective DNA double-strand break (DSB) repair, an accumulation of damaged DNA, and hypersensitivity of cells to genotoxic insults. Interestingly, we demonstrated that ECT2 promotes DNA repair and genome integrity largely independently of its canonical guanine nucleotide exchange activity. Together, these results suggest that ECT2 is directly involved in DSB repair and is an important genome caretaker.
    Keywords:  DNA damage; DNA damage response (DDR); DNA repair; double-strand break (DSB); genomic instability; guanine nucleotide exchange factor (GEF)
    DOI:  https://doi.org/10.1016/j.jbc.2021.101036
  5. Curr Opin Genet Dev. 2021 Jul 29. pii: S0959-437X(21)00093-9. [Epub ahead of print]71 114-119
    Advances in SUMO-based regulation of homologous recombination.
    Nalini Dhingra, Xiaolan Zhao.
      Homologous Recombination (HR) is a critical DNA repair mechanism for a range of genome lesions. HR is responsible for mending DNA double strand breaks (DSBs) using intact template DNA. In addition, many HR proteins help cope with DNA lesions generated from DNA replication and telomere deficiency. The functions of HR proteins are often regulated by protein modifications that can quickly and reversibly adjust substrate proteins' attributes. Sumoylation is one of the prevalent modifications that affects all steps of the HR processes and exerts diverse regulation on substrates. This review aims to summarize the most recent advances in our understanding of SUMO-based HR regulation and highlight some key questions that remain to be elucidated.
    DOI:  https://doi.org/10.1016/j.gde.2021.07.007
  6. J Biol Chem. 2021 Jul 30. pii: S0021-9258(21)00828-0. [Epub ahead of print] 101026
    The DNA-binding protein CST associates with the cohesin complex and promotes chromosome cohesion.
    P Logan Schuck, Lauren E Ball, Jason A Stewart.
      Sister chromatid cohesion (SCC), the pairing of sister chromatids following DNA replication until mitosis, is established by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. However, how SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress may lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST associates with the cohesin complex. Unexpectedly, we did not find evidence of altered cohesion or mitotic progression in the absence of CST; however, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we hypothesized that CST may be required to maintain or remodel SCC following DNA replication fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.
    Keywords:  CST; CTC1; Chromosome cohesion; DNA replication; STN1; TEN1; cohesin; fork stalling
    DOI:  https://doi.org/10.1016/j.jbc.2021.101026
  7. J Fungi (Basel). 2021 Jul 16. pii: 566. [Epub ahead of print]7(7):
    Functional Roles of Homologous Recombination and Non-Homologous End Joining in DNA Damage Response and Microevolution in Cryptococcus neoformans.
    Kwang-Woo Jung, Jong-Hyun Jung, Ha-Young Park.
      DNA double-strand breaks (DSBs) are the most deleterious type of DNA lesions because they cause loss of genetic information if not properly repaired. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are required for DSB repair. However, the relationship of HR and NHEJ in DNA damage stress is unknown in the radiation-resistant fungus Cryptococcus neoformans. In this study, we found that the expression levels of HR- and NHEJ-related genes were highly induced in a Rad53-Bdr1 pathway-dependent manner under genotoxic stress. Deletion of RAD51, which is one of the main components in the HR, resulted in growth under diverse types of DNA damage stress, whereas perturbations of KU70 and KU80, which belong to the NHEJ system, did not affect the genotoxic stresses except when bleomycin was used for treatment. Furthermore, deletion of both RAD51 and KU70/80 renders cells susceptible to oxidative stress. Notably, we found that deletion of RAD51 induced a hypermutator phenotype in the fluctuation assay. In contrast to the fluctuation assay, perturbation of KU70 or KU80 induced rapid microevolution similar to that induced by the deletion of RAD51. Collectively, Rad51-mediated HR and Ku70/Ku80-mediated NHEJ regulate the DNA damage response and maintain genome stability.
    Keywords:  Cryptococcus; DNA damage; homologous recombination; microevolution; non-homologous end joining
    DOI:  https://doi.org/10.3390/jof7070566
  8. Nat Cell Biol. 2021 Aug 05.
    ASTE1 promotes shieldin-complex-mediated DNA repair by attenuating end resection.
    Fei Zhao, Wootae Kim, Huanyao Gao, Chao Liu, Yong Zhang, Yuping Chen, Min Deng, Qin Zhou, Jinzhou Huang, Qi Hu, Shih-Hsun Chen, Somaira Nowsheen, Jake A Kloeber, Bo Qin, Ping Yin, Xinyi Tu, Guijie Guo, Sisi Qin, Chao Zhang, Ming Gao, Kuntian Luo, Yilun Liu, Zhenkun Lou, Jian Yuan.
      The shieldin complex functions as the downstream effector of 53BP1-RIF1 to promote DNA double-strand break end-joining by restricting end resection. The SHLD2 subunit binds to single-stranded DNA ends and blocks end resection through OB-fold domains. Besides blocking end resection, it is unclear how the shieldin complex processes SHLD2-bound single-stranded DNA and promotes non-homologous end-joining. Here, we identify a downstream effector of the shieldin complex, ASTE1, as a structure-specific DNA endonuclease that specifically cleaves single-stranded DNA and 3' overhang DNA. ASTE1 localizes to DNA damage sites in a shieldin-dependent manner. Loss of ASTE1 impairs non-homologous end-joining, leads to hyper-resection and causes defective immunoglobulin class switch recombination. ASTE1 deficiency also causes resistance to poly(ADP-ribose) polymerase inhibitors in BRCA1-deficient cells owing to restoration of homologous recombination. These findings suggest that ASTE1-mediated 3' single-stranded DNA end cleavage contributes to the control of DSB repair choice by 53BP1, RIF1 and shieldin.
    DOI:  https://doi.org/10.1038/s41556-021-00723-9
  9. Sci Adv. 2021 Aug;pii: eabe9254. [Epub ahead of print]7(32):
    GRB2 enforces homology-directed repair initiation by MRE11.
    Zu Ye, Shengfeng Xu, Yin Shi, Albino Bacolla, Aleem Syed, Davide Moiani, Chi-Lin Tsai, Qiang Shen, Guang Peng, Paul G Leonard, Darin E Jones, Bin Wang, John A Tainer, Zamal Ahmed.
      DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.
    DOI:  https://doi.org/10.1126/sciadv.abe9254
  10. EMBO J. 2021 Aug 04. e107413
    Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway.
    Julio C Y Liu, Ulrike Kühbacher, Nicolai B Larsen, Nikoline Borgermann, Dimitriya H Garvanska, Ivo A Hendriks, Leena Ackermann, Peter Haahr, Irene Gallina, Claire Guérillon, Emma Branigan, Ronald T Hay, Yoshiaki Azuma, Michael Lund Nielsen, Julien P Duxin, Niels Mailand.
      DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.
    Keywords:  DNA repair; DNA-protein crosslinks; SUMO; genome stability; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2020107413
  11. J Biol Chem. 2021 Jul 30. pii: S0021-9258(21)00827-9. [Epub ahead of print] 101025
    The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair.
    Qun Tang, Melike Çağlayan.
      The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffold protein, assembles multi-protein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein-protein interactions and DNA binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and hand off process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multi-protein/DNA complexes for efficient BER.
    Keywords:  Base excision repair; DNA ligase IIIα; DNA polymerase β; X-ray cross-complementing protein 1
    DOI:  https://doi.org/10.1016/j.jbc.2021.101025
  12. Curr Opin Genet Dev. 2021 Jul 30. pii: S0959-437X(21)00091-5. [Epub ahead of print]71 106-113
    Serine ADP-ribosylation in DNA-damage response regulation.
    Luca Palazzo, Marcin J Suskiewicz, Ivan Ahel.
      PARP1 and PARP2 govern the DNA-damage response by catalysing the reversible post-translational modification ADP-ribosylation. During the repair of DNA lesions, PARP1 and PARP2 combine with an accessory factor HPF1, which is required for the modification of target proteins on serine residues. Although the physiological role of individual ADP-ribosylation sites is still unclear, serine ADP-ribosylation at damage sites leads to the recruitment of chromatin remodellers and repair factors to ensure efficient DNA repair. ADP-ribosylation signalling is tightly controlled by the coordinated activities of (ADP-ribosyl)glycohydrolases PARG and ARH3 that, by reversing the modification, guarantee proper kinetics of DNA repair and cell cycle re-entry. The recent advances in the structural and mechanistic understanding of ADP-ribosylation provide new insights into human physiopathology and cancer therapy.
    DOI:  https://doi.org/10.1016/j.gde.2021.07.005
  13. Cancer Drug Resist. 2021 ;4 244-263
    Exploiting DNA repair pathways for tumor sensitization, mitigation of resistance, and normal tissue protection in radiotherapy.
    Jac A Nickoloff, Lynn Taylor, Neelam Sharma, Takamitsu A Kato.
      More than half of cancer patients are treated with radiotherapy, which kills tumor cells by directly and indirectly inducing DNA damage, including cytotoxic DNA double-strand breaks (DSBs). Tumor cells respond to these threats by activating a complex signaling network termed the DNA damage response (DDR). The DDR arrests the cell cycle, upregulates DNA repair, and triggers apoptosis when damage is excessive. The DDR signaling and DNA repair pathways are fertile terrain for therapeutic intervention. This review highlights strategies to improve therapeutic gain by targeting DDR and DNA repair pathways to radiosensitize tumor cells, overcome intrinsic and acquired tumor radioresistance, and protect normal tissue. Many biological and environmental factors determine tumor and normal cell responses to ionizing radiation and genotoxic chemotherapeutics. These include cell type and cell cycle phase distribution; tissue/tumor microenvironment and oxygen levels; DNA damage load and quality; DNA repair capacity; and susceptibility to apoptosis or other active or passive cell death pathways. We provide an overview of radiobiological parameters associated with X-ray, proton, and carbon ion radiotherapy; DNA repair and DNA damage signaling pathways; and other factors that regulate tumor and normal cell responses to radiation. We then focus on recent studies exploiting DSB repair pathways to enhance radiotherapy therapeutic gain.
    Keywords:  DNA double-strand break repair; DNA repair; cancer therapy; homologous recombination; non-homologous end-joining; radioprotection; radiosensitization
    DOI:  https://doi.org/10.20517/cdr.2020.89
  14. Biomolecules. 2021 Jul 20. pii: 1060. [Epub ahead of print]11(7):
    Intrinsic Disorder and Phosphorylation in BRCA2 Facilitate Tight Regulation of Multiple Conserved Binding Events.
    Manon Julien, Rania Ghouil, Ambre Petitalot, Sandrine M Caputo, Aura Carreira, Sophie Zinn-Justin.
      The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk.
    Keywords:  3D structure; DNA repair; NMR; cancer; disorder; meiosis; mitosis; phosphorylation; protein-protein interaction; variants
    DOI:  https://doi.org/10.3390/biom11071060
  15. DNA Repair (Amst). 2021 Jul 26. pii: S1568-7864(21)00149-X. [Epub ahead of print]106 103193
    Nonhomologous DNA end joining of nucleosomal substrates in a purified system.
    Christina A Gerodimos, Go Watanabe, Michael R Lieber.
      The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.
    Keywords:  Artemis; DNA damage; DNA recombination; DNA repair; DNA-dependent serine/threonine protein kinase (DNA-PK); Double-strand DNA breaks; Endonuclease; Ligase; Nonhomologous DNA end joining; Nucleosome
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103193
  16. Cancers (Basel). 2021 Jul 29. pii: 3819. [Epub ahead of print]13(15):
    Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies.
    Caroline Molinaro, Alain Martoriati, Katia Cailliau.
      Cells respond to genotoxic stress through a series of complex protein pathways called DNA damage response (DDR). These monitoring mechanisms ensure the maintenance and the transfer of a correct genome to daughter cells through a selection of DNA repair, cell cycle regulation, and programmed cell death processes. Canonical or non-canonical DDRs are highly organized and controlled to play crucial roles in genome stability and diversity. When altered or mutated, the proteins in these complex networks lead to many diseases that share common features, and to tumor formation. In recent years, technological advances have made it possible to benefit from the principles and mechanisms of DDR to target and eliminate cancer cells. These new types of treatments are adapted to the different types of tumor sensitivity and could benefit from a combination of therapies to ensure maximal efficiency.
    Keywords:  DDR inhibitors; DNA damage response; DNA damage therapy; DNA repair; cancers; cell cycle
    DOI:  https://doi.org/10.3390/cancers13153819
  17. Cancers (Basel). 2021 Jul 28. pii: 3790. [Epub ahead of print]13(15):
    Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells.
    Gro Elise Rødland, Sissel Hauge, Grete Hasvold, Lilli T E Bay, Tine T H Raabe, Mrinal Joel, Randi G Syljuåsen.
      Inhibitors of WEE1 and ATR kinases are considered promising for cancer treatment, either as monotherapy or in combination with chemo- or radiotherapy. Here, we addressed whether simultaneous inhibition of WEE1 and ATR might be advantageous. Effects of the WEE1 inhibitor MK1775 and ATR inhibitor VE822 were investigated in U2OS osteosarcoma cells and in four lung cancer cell lines, H460, A549, H1975, and SW900, with different sensitivities to the WEE1 inhibitor. Despite the differences in cytotoxic effects, the WEE1 inhibitor reduced the inhibitory phosphorylation of CDK, leading to increased CDK activity accompanied by ATR activation in all cell lines. However, combining ATR inhibition with WEE1 inhibition could not fully compensate for cell resistance to the WEE1 inhibitor and reduced cell viability to a variable extent. The decreased cell viability upon the combined treatment correlated with a synergistic induction of DNA damage in S-phase in U2OS cells but not in the lung cancer cells. Moreover, less synergy was found between ATR and WEE1 inhibitors upon co-treatment with radiation, suggesting that single inhibitors may be preferable together with radiotherapy. Altogether, our results support that combining WEE1 and ATR inhibitors may be beneficial for cancer treatment in some cases, but also highlight that the effects vary between cancer cell lines.
    Keywords:  ATR kinase; CDK activity; WEE1 kinase; combination therapy; lung cancer; radiosensitization; replication stress; synergy
    DOI:  https://doi.org/10.3390/cancers13153790
  18. Cancer Res Treat. 2021 Aug 06.
    Inhibition of WEE1 Potentiates Sensitivity to PARP Inhibitor in Biliary Tract Cancer.
    Hye-Rim Seo, Ah-Rong Nam, Ju-Hee Bang, Kyoung-Seok Oh, Jae-Min Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh.
      Purpose: Up to 20% of patients with biliary tract cancer (BTC) have alterations in DNA damage response (DDR) genes, including homologous recombination (HR) genes. Therefore, the DDR pathway could be a promising target for new drug development in BTC. We aim to investigate the anti-tumor effects using PARP and WEE1 inhibitors in BTC.Materials and Methods: We used 10 biliary tract cancer cell lines to evaluate an anti-tumor effect of olaparib (a PARP inhibitor) and AZD1775 (a WEE1 inhibitor) in in vitro. Additionally, we established SNU869 xenograft model for in vivo experiments.
    Results: In this study, we observed a modest anti-proliferative effect of olaparib. DNA double-strand break (DSB) and apoptosis were increased by olaparib in BTC cells. However, olaparib-induced DNA DSB was repaired through the HR pathway, and G2 arrest was induced to secure the time for repair. As AZD1775 typically regulates the G2/M checkpoint, we combined olaparib with AZD1775 to abrogate G2 arrest. We observed that AZD1775 downregulated p-CDK1, a G2/M cell cycle checkpoint protein, and induced early mitotic entry. AZD1775 also decreased CtIP and RAD51 expression and disrupted HR repair. In xenograft model, olaparib plus AZD1775 treatment reduced tumor growth more potently than did monotherapy with either drug.
    Conclusion: This is the first study to suggest that olaparib combined with AZD1775 can induce synergistic anti-tumor effects against BTC. Combination therapy that blocks dual PARP and WEE1 has the potential to be further clinically developed for BTC patients.
    Keywords:  Biliary tract cancer; Cell cycle checkpoint; DNA damage response; Homologous recombination; PARP; WEE1
    DOI:  https://doi.org/10.4143/crt.2021.473
  19. Biochim Biophys Acta Rev Cancer. 2021 Jul 28. pii: S0304-419X(21)00094-9. [Epub ahead of print]1876(2): 188597
    Inhibiting homologous recombination by targeting RAD51 protein.
    A Demeyer, H Benhelli-Mokrani, B Chénais, P Weigel, F Fleury.
      Homologous recombination (HR) is involved in repairing DNA double-strand breaks (DSB), the most harmful for the cell. Regulating HR is essential for maintaining genomic stability. In many forms of cancer, overactivation of HR increases tumor resistance to DNA-damaging treatments. RAD51, HR's core protein, is very often over-expressed in these cancers and plays a critical role in cancer cell development and survival. Targeting RAD51 directly to reduce its activity and its expression is therefore one strategy to sensitize and overcome resistance cancer cells to existing DNA-damaging therapies which remains the limiting factor for the success of targeted therapy. This review describes the structure and biological roles of RAD51, summarizes the different targeted sites of RAD51 and its inhibitory compounds discovered and described in the last decade.
    Keywords:  Cancer sensitization; DNA repair; Inhibitor; RAD51; Small molecule
    DOI:  https://doi.org/10.1016/j.bbcan.2021.188597
  20. Nat Commun. 2021 Aug 06. 12(1): 4750
    Dpb4 promotes resection of DNA double-strand breaks and checkpoint activation by acting in two different protein complexes.
    Erika Casari, Elisa Gobbini, Marco Gnugnoli, Marco Mangiagalli, Michela Clerici, Maria Pia Longhese.
      Budding yeast Dpb4 (POLE3/CHRAC17 in mammals) is a highly conserved histone fold protein that is shared by two protein complexes: the chromatin remodeler ISW2/hCHRAC and the DNA polymerase ε (Pol ε) holoenzyme. In Saccharomyces cerevisiae, Dpb4 forms histone-like dimers with Dls1 in the ISW2 complex and with Dpb3 in the Pol ε complex. Here, we show that Dpb4 plays two functions in sensing and processing DNA double-strand breaks (DSBs). Dpb4 promotes histone removal and DSB resection by interacting with Dls1 to facilitate the association of the Isw2 ATPase to DSBs. Furthermore, it promotes checkpoint activation by interacting with Dpb3 to facilitate the association of the checkpoint protein Rad9 to DSBs. Persistence of both Isw2 and Rad9 at DSBs is enhanced by the A62S mutation that is located in the Dpb4 histone fold domain and increases Dpb4 association at DSBs. Thus, Dpb4 exerts two distinct functions at DSBs depending on its interactors.
    DOI:  https://doi.org/10.1038/s41467-021-25090-9
  21. DNA Repair (Amst). 2021 Jul 26. pii: S1568-7864(21)00148-8. [Epub ahead of print]106 103192
    Dealing with transcription-blocking DNA damage: Repair mechanisms, RNA polymerase II processing and human disorders.
    Nan Jia, Chaowan Guo, Yuka Nakazawa, Diana van den Heuvel, Martijn S Luijsterburg, Tomoo Ogi.
      Transcription-blocking DNA lesions (TBLs) in genomic DNA are triggered by a wide variety of DNA-damaging agents. Such lesions cause stalling of elongating RNA polymerase II (RNA Pol II) enzymes and fully block transcription when unresolved. The toxic impact of DNA damage on transcription progression is commonly referred to as transcription stress. In response to RNA Pol II stalling, cells activate and employ transcription-coupled repair (TCR) machineries to repair cytotoxic TBLs and resume transcription. Increasing evidence indicates that the modification and processing of stalled RNA Pol II is an integral component of the cellular response to and the repair of TBLs. If TCR pathways fail, the prolonged stalling of RNA Pol II will impede global replication and transcription as well as block the access of other DNA repair pathways that may act upon the TBL. Consequently, such prolonged stalling will trigger profound genome instability and devastating clinical features. In this review, we will discuss the mechanisms by which various types of TBLs are repaired by distinct TCR pathways and how RNA Pol II processing is regulated during these processes. We will also discuss the clinical consequences of transcription stress and genotype-phenotype correlations of related TCR-deficiency disorders.
    Keywords:  Ageing-associated symptoms; Nucleotide excision repair; Transcription-blocking DNA lesions; Transcription-coupled repair; Ubiquitination of RNA polymerase II
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103192
  22. Int J Mol Sci. 2021 Jul 30. pii: 8199. [Epub ahead of print]22(15):
    Targeting DNA Damage Response and Repair to Enhance Therapeutic Index in Cisplatin-Based Cancer Treatment.
    Robert Csaba Kiss, Fen Xia, Scarlett Acklin.
      Platinum-based chemotherapies, such as cisplatin, play a large role in cancer treatment. The development of resistance and treatment toxicity creates substantial barriers to disease control, yet. To enhance the therapeutic index of cisplatin-based chemotherapy, it is imperative to circumvent resistance and toxicity while optimizing tumor sensitization. One of the primary mechanisms by which cancer cells develop resistance to cisplatin is through upregulation of DNA repair pathways. In this review, we discuss the DNA damage response in the context of cisplatin-induced DNA damage. We describe the proteins involved in the pathways and their roles in resistance development. Common biomarkers for cisplatin resistance and their utilization to improve patient risk stratification and treatment personalization are addressed. Finally, we discuss some of the current treatments and future strategies to circumvent the development of cisplatin resistance.
    Keywords:  DNA damage response; anticancer drugs; cisplatin; cisplatin resistance
    DOI:  https://doi.org/10.3390/ijms22158199
  23. Genes (Basel). 2021 Jul 02. pii: 1034. [Epub ahead of print]12(7):
    Understanding BRCA2 Function as a Tumor Suppressor Based on Domain-Specific Activities in DNA Damage Responses.
    Paul R Andreassen, Joonbae Seo, Constanze Wiek, Helmut Hanenberg.
      BRCA2 is an essential genome stability gene that has various functions in cells, including roles in homologous recombination, G2 checkpoint control, protection of stalled replication forks, and promotion of cellular resistance to numerous types of DNA damage. Heterozygous mutation of BRCA2 is associated with an increased risk of developing cancers of the breast, ovaries, pancreas, and other sites, thus BRCA2 acts as a classic tumor suppressor gene. However, understanding BRCA2 function as a tumor suppressor is severely limited by the fact that ~70% of the encoded protein has not been tested or assigned a function in the cellular DNA damage response. Remarkably, even the specific role(s) of many known domains in BRCA2 are not well characterized, predominantly because stable expression of the very large BRCA2 protein in cells, for experimental purposes, is challenging. Here, we review what is known about these domains and the assay systems that are available to study the cellular roles of BRCA2 domains in DNA damage responses. We also list criteria for better testing systems because, ultimately, functional assays for assessing the impact of germline and acquired mutations identified in genetic screens are important for guiding cancer prevention measures and for tailored cancer treatments.
    Keywords:  BRCA2; DNA binding; DNA damage responses; DNA repair; G2 checkpoint; homologous recombination; tumor suppressor; variants of uncertain significance
    DOI:  https://doi.org/10.3390/genes12071034
  24. DNA Repair (Amst). 2021 Jul 29. pii: S1568-7864(21)00153-1. [Epub ahead of print]107 103197
    Non-canonical function of nuclear PTEN and its implication on tumorigenesis.
    Sandip Misra, Ginia Ghosh, Sougata Ghosh Chowdhury, Parimal Karmakar.
      Suppression of genomic instability is the key to prevent tumor development. PTEN is a unique tumor suppressor protein having both lipid and protein phosphatase activities. Interestingly though it is a cytoplasmic protein, but a significant pool of PTEN can also be localized in nucleus. The function of cytoplasmic PTEN is well defined and extensively studied in various literatures focusing mainly on the negative regulation of oncogenic PI-3Kinase-AKT pathway but functional regulation of nuclear PTEN is less defined and therefore it is a fascinating subject of research in cancer biology. Post-translation modulation of PTEN such as phosphorylation, sumorylation, acetylation and methylation also regulates its cellular localization, protein-protein association and catalytic function. Loss or mutation in PTEN is associated with the development of tumors in various tissues from the brain to prostate. Here we have summarized the role of nuclear PTEN and its epigenetic modulation in various DNA metabolic pathways, for example, DNA damage response, DNA repair, DNA replication, DNA segregation etc. Further, pathways involved in nuclear PTEN degradation are also discussed. Additionally, we also emphasize probable potential targets associated with PTEN pathway for chemotherapeutic purpose.
    Keywords:  Chromatin; DNA repair; PTEN Post translational modification; Replication stress
    DOI:  https://doi.org/10.1016/j.dnarep.2021.103197
  25. Int J Mol Sci. 2021 Jul 21. pii: 7782. [Epub ahead of print]22(15):
    Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.
    Anudari Munkhjargal, Myung-Jin Kim, Da-Yeon Kim, Young-Jun Jeon, Young-Hoon Kee, Lark-Kyun Kim, Yong-Hwan Kim.
      Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.
    Keywords:  CHK1 inhibitors; Fanconi anemia; PML nuclear body; interstrand DNA crosslink
    DOI:  https://doi.org/10.3390/ijms22157782
  26. Mol Cell. 2021 Jul 27. pii: S1097-2765(21)00557-8. [Epub ahead of print]
    Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.
    Amanda K Chaplin, Steven W Hardwick, Antonia Kefala Stavridi, Christopher J Buehl, Noah J Goff, Virginie Ropars, Shikang Liang, Taiana Maia De Oliveira, Dimitri Y Chirgadze, Katheryn Meek, Jean-Baptiste Charbonnier, Tom L Blundell.
      Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.
    Keywords:  DNA repair; DNA-PK; NHEJ; cryo-EM; long-range synaptic complexes; non-homologous end joining
    DOI:  https://doi.org/10.1016/j.molcel.2021.07.005
  27. Blood Cancer J. 2021 Jul 31. 11(7): 137
    High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.
    Ruochen Jia, Leon Kutzner, Anna Koren, Kathrin Runggatscher, Peter Májek, André C Müller, Michael Schuster, Christoph Bock, Joanna I Loizou, Stefan Kubicek, Robert Kralovics.
      Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.
    DOI:  https://doi.org/10.1038/s41408-021-00531-2
  28. Elife. 2021 Aug 03. pii: e59828. [Epub ahead of print]10
    Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.
    Knut H Lauritzen, Maria Belland Olsen, Mohammed Shakil Ahmed, Kuan Yang, Johanne Egge Rinholm, Linda H Bergersen, Qin Ying Esbensen, Lars Jansen Sverkeli, Mathias Ziegler, Håvard Attramadal, Bente Halvorsen, Pål Aukrust, Arne Yndestad.
      Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.
    Keywords:  DNA repair; NAD+; SIRT3; biochemistry; cardiovascular disease; chemical biology; human; mitochondrial dna; mouse; nicotinamide riboside
    DOI:  https://doi.org/10.7554/eLife.59828
  29. Front Oncol. 2021 ;11 706337
    DNA Polymerase Iota Promotes Esophageal Squamous Cell Carcinoma Proliferation Through Erk-OGT-Induced G6PD Overactivation.
    Zhenzi Su, Aidi Gao, Xiaoqing Li, Shitao Zou, Chao He, Jinchang Wu, Wei-Qun Ding, Jundong Zhou.
      Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers with rapid progression and a high mortality rate. Our previous study demonstrated that DNA polymerase iota (Pol ι) is overexpressed in ESCC tumors and correlates with poor prognosis. However, its role in ESCC proliferation remains obscure. We report here that Pol ι promotes ESCC proliferation and progression through Erk- O-GlcNAc transferase (OGT) regulated Glucose-6-phosphate dehydrogenase (G6PD) overactivation. Cell clonogenic ability was assessed by colony formation assay. Cell proliferation was assessed by EdU incorporation assay. Our transcriptome data was reanalyzed by GSEA and validated by analysis of cellular metabolism, G6PD activity, and cellular NADPH concentration. The level of Pol ι, OGT, G6PD and O-GlcNAcylation in ESCC cells and patient samples were analyzed. The MEK inhibitor PD98059 was applied to confirm OGT expression regulation by the Erk signaling. The G6PD inhibitor polydatin was used to examine the role of G6PD activation in Pol ι promoted proliferation. We found that Pol ι promotes ESCC proliferation. It shunted the glucose flux towards the pentose phosphate pathway (PPP) by activating G6PD through OGT-promoted O-GlcNAcylation. The expression of OGT was positively correlated with Pol ι expression and O-GlcNAcylation. Notably, elevated O-GlcNAcylation was correlated with poor prognosis in ESCC patients. Pol ι was shown to stimulate Erk signaling to enhance OGT expression, and the G6PD inhibitor polydatin attenuated Pol ι induced tumor growth in vitro and in vivo. In conclusion, Pol ι activates G6PD through Erk-OGT-induced O-GlcNAcylation to promote the proliferation and progression of ESCC, supporting the notion that Pol ι is a potential biomarker and therapeutic target of ESCC.
    Keywords:  DNA polymerase iota; ESCC; Erk signaling pathway; G6PD activity; tumor proliferation
    DOI:  https://doi.org/10.3389/fonc.2021.706337
  30. Cell Rep. 2021 Aug 03. pii: S2211-1247(21)00905-0. [Epub ahead of print]36(5): 109478
    BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.
    Xavier Renaudin, Miyoung Lee, Mona Shehata, Eva-Maria Surmann, Ashok R Venkitaraman.
      Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation. In BRCA2-deficient cells, intracellular reactive oxygen species (ROS) are elevated, and ROS scavengers suppress the mtDNA defects. Conversely, wild-type cells exposed to oxidative stress by pharmacologic or genetic manipulation phenocopy these defects. Mechanistically, we find that 8-oxoguanine accumulation in mtDNA caused by oxidative stress suffices to impair recruitment of the mitochondrial enzyme RNaseH1 to sites of R-loop accrual, restricting mtDNA replication initiation. Thus, oxidative stress impairs RNaseH1 function to cripple mtDNA maintenance. Our findings highlight a molecular mechanism that links oxidative stress to mitochondrial dysfunction and is elicited by the inactivation of genes implicated in neurodegeneration and cancer.
    Keywords:  BRCA2; PRPF8; R-loops; RNaseH1; SETX; cancer; mitochondrial DNA replication; neurodegeneration; oxidative stress
    DOI:  https://doi.org/10.1016/j.celrep.2021.109478
  31. Proc Natl Acad Sci U S A. 2021 Aug 10. pii: e2021998118. [Epub ahead of print]118(32):
    The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.
    Osama Garwain, Xiaoming Sun, Divya Ramalingam Iyer, Rui Li, Lihua Julie Zhu, Paul D Kaufman.
      Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.
    Keywords:  DNA damage; chromatin; mitosis
    DOI:  https://doi.org/10.1073/pnas.2021998118
  32. Oncogene. 2021 Aug 04.
    Wild-type IDH2 protects nuclear DNA from oxidative damage and is a potential therapeutic target in colorectal cancer.
    Shuang Qiao, Wenhua Lu, Christophe Glorieux, Jiangjiang Li, Peiting Zeng, Ning Meng, Huiqin Zhang, Shijun Wen, Peng Huang.
      Although the role of isocitrate dehydrogenase (IDH) mutation in promoting cancer development has been well-characterized, the impact of wild-type IDH on cancer cells remains unclear. Here we show that the wild-type isocitrate dehydrogenase 2 (IDH2) is highly expressed in colorectal cancer (CRC) cells, and plays an unexpected role in protecting the cancer cells from oxidative damage. Genetic abrogation of IDH2 in CRC cells leads to reactive oxygen species (ROS)-mediated DNA damage and an accumulation of 8-oxoguanine with DNA strand breaks, which activates DNA damage response (DDR) with elevated γH2AX and phosphorylation of ataxia telangiectasia-mutated (ATM) protein, leading to a partial cell cycle arrest and eventually cell senescence. Mechanistically, the suppression of IDH2 results in a reduction of the tricarboxylic acid (TCA) cycle activity due to a decrease in the conversion of isocitrate to α-ketoglutarate (α-KG) with a concurrent decrease in NADPH production, leading to ROS accumulation and oxidative DNA damage. Importantly, abrogation of IDH2 inhibits CRC cell growth in vitro and in vivo, and renders CRC cells more vulnerable to DNA-damaging drugs. Screening of an FDA-approved drug library has identified oxaliplatin as a compound highly effective against CRC cells when IDH2 was suppressed. Our study has uncovered an important role of the wild-type IDH2 in protecting DNA from oxidative damage, and provides a novel biochemical basis for developing metabolic intervention strategy for cancer treatment.
    DOI:  https://doi.org/10.1038/s41388-021-01968-2
  33. Blood. 2021 Aug 25. pii: blood.2021012629. [Epub ahead of print]
    Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.
    Jan-Erik Messling, Karl Agger, Kasper L Andersen, Kristina Kromer, Hanna Maria Kuepper, Anders H Lund, Kristian Helin.
      Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed as current treatments do not cure the majority of AML patients. Here, we report on a domain-focused, kinome-wide CRISPR-Cas9 screen to identify protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. We show that loss of RIOK2 leads to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, we demonstrate that the ATPase function of RIOK2 is required for cell survival. By using a small molecule inhibitor, we show that pharmacological inhibition of RIOK2 similarly leads to loss of protein synthesis and apoptosis and affects leukemic cell growth in vivo. Our results provide proof-of-concept for targeting RIOK2 as a potential treatment for AML patients.
    DOI:  https://doi.org/10.1182/blood.2021012629
  34. EMBO Rep. 2021 Aug 04. e52145
    Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.
    Abby M Green, Rachel A DeWeerd, David R O'Leary, Ava R Hansen, Katharina E Hayer, Katarzyna Kulej, Ariel S Dineen, Julia H Szeto, Benjamin A Garcia, Matthew D Weitzman.
      The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.
    Keywords:  APOBEC3A; CCT chaperonin; cytidine deaminase; mutational signatures; protein interaction
    DOI:  https://doi.org/10.15252/embr.202052145
  35. Genes (Basel). 2021 Jul 19. pii: 1096. [Epub ahead of print]12(7):
    Hydroxyurea-The Good, the Bad and the Ugly.
    Marcelina W Musiałek, Dorota Rybaczek.
      Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and-recently-a promising factor in preventing cognitive decline in Alzheimer's disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.
    Keywords:  DNA replication checkpoint; cell cycle arrest; hydroxyurea; replication stress; ribonucleotide reductase
    DOI:  https://doi.org/10.3390/genes12071096
  36. Breast Cancer. 2021 Aug 03.
    Molecular targeted drugs resistance impairs double-strand break repair and sensitizes ER-positive breast cancer to PARP inhibitors.
    Yuna Suzuki, Wu Wenwen, Tomohiko Ohta, Shin-Ichi Hayashi.
      BACKGROUND: There are various treatments for estrogen-positive breast cancer, mainly hormone therapy and molecular-targeted drugs. Acquiring resistance to these drugs is a major clinical problem. Additionally, little is known about the effect of drug resistance on the DNA repair mechanism. Poly ADP ribose polymerase (PARP) inhibitors currently used for treating HER2-negative metastatic breast cancer with BRCA mutations have been shown to be effective in BRCA-deficient cells with impaired homologous recombination repair. Here, we investigated the effect of drug resistance acquisition on the DNA repair mechanism and the effect of PARP inhibitors on ER (estrogen receptor) -positive breast cancer.METHODS: We investigated changes in the expression of DNA repair mechanism-related factors and repair ability of double-strand breaks (DSB) in various drug-resistant cell lines established in our laboratory. Additionally, PARP inhibitor susceptibility was investigated using olaparib.
    RESULTS: DSB repairs in MCF-7 and hormone therapy-resistant model cells were normal, and these cells demonstrated low sensitivity to olaparib. The resistant cell lines against CDK4/6 inhibitors, fulvestrant and mTOR/PI3K inhibitors showed decreased DSB repair ability and high olaparib sensitivity. They showed low sensitivity to CDK4/6 inhibitors, a close link between acquiring resistance to CDK4/6 inhibitors and hypersensitivity to olaparib.
    CONCLUSIONS: Our study suggests some cases of acquiring drug resistance impairs DSB repair ability and sensitizes ER-positive breast cancer to PARP inhibitors.
    Keywords:  Breast cancer; CDK4/6 inhibitor; DNA repair; Drug resistance; PARP inhibitor
    DOI:  https://doi.org/10.1007/s12282-021-01282-5
  37. Mol Cell. 2021 Aug 05. pii: S1097-2765(21)00584-0. [Epub ahead of print]81(15): 3227
    Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.
    Ke Cong, Min Peng, Arne Nedergaard Kousholt, Wei Ting C Lee, Silviana Lee, Sumeet Nayak, John Krais, Pamela S VanderVere-Carozza, Katherine S Pawelczak, Jennifer Calvo, Nicholas J Panzarino, John J Turchi, Neil Johnson, Jos Jonkers, Eli Rothenberg, Sharon B Cantor.
      
    DOI:  https://doi.org/10.1016/j.molcel.2021.07.015
  38. Dev Cell. 2021 Aug 02. pii: S1534-5807(21)00592-X. [Epub ahead of print]
    Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies.
    Devon A Lukow, Erin L Sausville, Pavit Suri, Narendra Kumar Chunduri, Angela Wieland, Justin Leu, Joan C Smith, Vishruth Girish, Ankith A Kumar, Jude Kendall, Zihua Wang, Zuzana Storchova, Jason M Sheltzer.
      Aneuploidy is a ubiquitous feature of human tumors, but the acquisition of aneuploidy typically antagonizes cellular fitness. To investigate how aneuploidy could contribute to tumor growth, we triggered periods of chromosomal instability (CIN) in human cells and then exposed them to different culture environments. We discovered that transient CIN reproducibly accelerates the acquisition of resistance to anti-cancer therapies. Single-cell sequencing revealed that these resistant populations develop recurrent aneuploidies, and independently deriving one chromosome-loss event that was frequently observed in paclitaxel-resistant cells was sufficient to decrease paclitaxel sensitivity. Finally, we demonstrated that intrinsic levels of CIN correlate with poor responses to numerous therapies in human tumors. Our results show that, although CIN generally decreases cancer cell fitness, it also provides phenotypic plasticity to cancer cells that can allow them to adapt to diverse stressful environments. Moreover, our findings suggest that aneuploidy may function as an under-explored cause of therapy failure.
    Keywords:  CIN; aneuploidy; cancer; drug resistance; evolution
    DOI:  https://doi.org/10.1016/j.devcel.2021.07.009
  39. Cancers (Basel). 2021 Jul 21. pii: 3663. [Epub ahead of print]13(15):
    BRCA2 Promotes Spontaneous Homologous Recombination In Vivo.
    Adam D Brown, Scott Greenman, Alison B Claybon, Alexander J R Bishop.
      BACKGROUND: BRCA2 is known to be a tumor suppressor involved in homologous recombination repair and presumed to prevent genome instability in normal tissues prior to the development of tumors. Typical assessment of BRCA2 deficiency on the genome involves cell-based models using cancer cells with mixed genetic contexts, but the role in normal tissue in vivo has not been clearly demonstrated.METHODS: Using conditional deletion of Brca2 exon 11, the region containing all eight BRC repeats, in the retinal pigment epithelium and the pink-eyed unstable mouse model, we evaluate the frequency of DNA deletion events.
    RESULTS: In the current study, we show that conditional loss of Brca2 exon 11 results in a decreased frequency of spontaneous homologous recombination compared to wild-type mice. Of note, we observe no apparent concomitant increase in events that indicate single-strand annealing by the pink-eyed unstable mouse model.
    CONCLUSIONS: Therefore, our results demonstrate that BRCA2, as expected, is required for high-fidelity homologous recombination DNA repair in normal tissues, here in a tissue undergoing normal proliferation through normal development.
    Keywords:  BRCA2; homologous recombination; mouse; pink-eyed unstable
    DOI:  https://doi.org/10.3390/cancers13153663
  40. Nat Cell Biol. 2021 Aug 05.
    ASTE1 cutting to block DNA end resection.
    Yizhou Joseph He, Dipanjan Chowdhury.
      
    DOI:  https://doi.org/10.1038/s41556-021-00731-9
  41. Blood. 2021 Aug 06. pii: blood.2021012805. [Epub ahead of print]
    Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.
    Miguel A Galindo-Campos, Nura Lutfi, Sarah Bonnin, Carlos Martínez, Talia Velasco-Hernandez, Violeta García-Hernández, Juan Martin-Caballero, Coral Ampurdanés, Ramón Gimeno, Lluis Colomo, Gael Roue, Guillaume Guilbaud, Françoise Dantzer, Pilar Navarro, Matilde Murga, Oscar Fernandez-Capetillo, Anna Bigas, Pablo Menendez, Julian Sale, José Yélamos.
      Dysregulation of the c-Myc oncogene occurs in a wide variety of haematologic malignancies and its overexpression has been linked with aggressive tumour progression. Here, we show that Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphomas. PARP-1 and PARP-2 catalyse the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA-strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphomas, while PARP-1-deficiency accelerates lymphomagenesis in the Em-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in pre-leukemic Em-Myc B cells resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1-deficiency induces a proinflammatory response, and an increase in regulatory T cells likely contributing to immune escape of B-cell lymphomas, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centred therapeutic strategies with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumours.
    DOI:  https://doi.org/10.1182/blood.2021012805
  42. Biomolecules. 2021 Jul 03. pii: 981. [Epub ahead of print]11(7):
    Dual PARP and RAD51 Inhibitory Drug Conjugates Show Synergistic and Selective Effects on Breast Cancer Cells.
    Matthews M Malka, Julia Eberle, Kathrin Niedermayer, Darius P Zlotos, Lisa Wiesmüller.
      The genetic principle of synthetic lethality has most successfully been exploited in therapies engaging Poly-ADP-ribose-polymerase (PARP) inhibitors to treat patients with homologous recombination (HR)-defective tumors. In this work, we went a step further following the idea of a local molecular cooperation and designed hybrid compounds M1-M3. The drug conjugates M1-M3 combine Olaparib, the first PARP inhibitor approved for clinical use, with Cpd 1, an inhibitor of RAD51 that blocks its HR functions and yet permits RAD51 nucleoprotein filament formation on single-stranded DNA. While in M2 and M3, the parental drugs are linked by -CO-(CH2)n-CO-spacers (n = 2 and 4, respectively), they are directly merged omitting the piperazine ring of Olaparib in M1. Monitoring anti-survival effects of M1-M3 in six breast cancer cell lines of different molecular subtypes showed that in each cell line, at least one of the drug conjugates decreased viability by one to two orders of magnitude compared with parental drugs. While triple-negative breast cancer (TNBC) cells with frequent BRCA1 pathway dysfunction were sensitive to spacer-linked hybrid compounds M1 and M2 regardless of their HR capacities, non-TNBC cells were responsive to the merged drug conjugate M1 only, suggesting different spatial requirements for dual inhibition in these two groups of cell lines. These results demonstrate that, depending on chemical linkage, dual PARP1-RAD51 inhibitory drugs can either sensitize non-TNBC and re-sensitize TNBC cells, or discriminate between these groups of cells.
    Keywords:  Olaparib; PARP inhibitor; RAD51 inhibitor; anticancer drug hybrids; drug conjugates; triple-negative breast cancer
    DOI:  https://doi.org/10.3390/biom11070981
  43. Cancers (Basel). 2021 Jul 25. pii: 3733. [Epub ahead of print]13(15):
    Targeting Replication Stress Using CHK1 Inhibitor Promotes Innate and NKT Cell Immune Responses and Tumour Regression.
    Martina Proctor, Jazmina L Gonzalez Cruz, Sheena M Daignault-Mill, Margaret Veitch, Bijun Zeng, Anna Ehmann, Muhammed Sabdia, Cameron Snell, Colm Keane, Riccardo Dolcetti, Nikolas K Haass, James W Wells, Brian Gabrielli.
      Drugs selectively targeting replication stress have demonstrated significant preclinical activity, but this has not yet translated into an effective clinical treatment. Here we report that targeting increased replication stress with a combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea targets also promotes pro-inflammatory cytokine/chemokine expression that is independent of cGAS-STING pathway activation and immunogenic cell death in human and murine melanoma cells. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. It increases cytotoxic CD8+ T cell activity, but the major adaptive immune response is a pronounced NKT cell tumour infiltration. Treatment also promotes an immunosuppressive tumour microenvironment through CD4+ Treg and FoxP3+ NKT cells. The number of these accumulated during treatment, the increase in FoxP3+ NKT cells numbers correlates with the decrease in activated NKT cells, suggesting they are a consequence of the conversion of effector to suppressive NKT cells. Whereas tumour infiltrating CD8+ T cell PD-1 and tumour PD-L1 expression was increased with treatment, peripheral CD4+ and CD8+ T cells retained strong anti-tumour activity. Despite increased CD8+ T cell PD-1, combination with anti-PD-1 did not improve response, indicating that immunosuppression from Tregs and FoxP3+ NKT cells are major contributors to the immunosuppressive tumour microenvironment. This demonstrates that therapies targeting replication stress can be well tolerated, not adversely affect immune responses, and trigger an effective anti-tumour immune response.
    Keywords:  CHK1 inhibitor; NKT cells; adaptive immune response; immunogenic cell death; innate immune response; melanoma; replication stress
    DOI:  https://doi.org/10.3390/cancers13153733
  44. Cell Rep. 2021 Aug 03. pii: S2211-1247(21)00912-8. [Epub ahead of print]36(5): 109485
    SMC complexes are guarded by the SUMO protease Ulp2 against SUMO-chain-mediated turnover.
    Ivan Psakhye, Dana Branzei.
      Structural maintenance of chromosomes (SMCs) complexes, cohesin, condensin, and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5-deleted cells can also be bypassed by simultaneous loss of the proliferating cell nuclear antigen (PCNA) unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO chain assembly, which we propose to time the availability of SMC complexes on chromatin.
    Keywords:  Cohesin; Condensin; Pds5; SENP/Ulp proteases; SMC complexes; STUbL-mediated proteasomal degradation; SUMO; SUMO chains; Slx5/8; Smc5/6
    DOI:  https://doi.org/10.1016/j.celrep.2021.109485
  45. Nature. 2021 Aug 04.
    Genetic insights into biological mechanisms governing human ovarian ageing.
    Katherine S Ruth, Felix R Day, Jazib Hussain, Ana Martínez-Marchal, Catherine E Aiken, Ajuna Azad, Deborah J Thompson, Lucie Knoblochova, Hironori Abe, Jane L Tarry-Adkins, Javier Martin Gonzalez, Pierre Fontanillas, Annique Claringbould, Olivier B Bakker, Patrick Sulem, Robin G Walters, Chikashi Terao, Sandra Turon, Momoko Horikoshi, Kuang Lin, N Charlotte Onland-Moret, Aditya Sankar, Emil Peter Thrane Hertz, Pascal N Timshel, Vallari Shukla, Rehannah Borup, Kristina W Olsen, Paula Aguilera, Mònica Ferrer-Roda, Yan Huang, Stasa Stankovic, Paul R H J Timmers, Thomas U Ahearn, Behrooz Z Alizadeh, Elnaz Naderi, Irene L Andrulis, Alice M Arnold, Kristan J Aronson, Annelie Augustinsson, Stefania Bandinelli, Caterina M Barbieri, Robin N Beaumont, Heiko Becher, Matthias W Beckmann, Stefania Benonisdottir, Sven Bergmann, Murielle Bochud, Eric Boerwinkle, Stig E Bojesen, Manjeet K Bolla, Dorret I Boomsma, Nicholas Bowker, Jennifer A Brody, Linda Broer, Julie E Buring, Archie Campbell, Harry Campbell, Jose E Castelao, Eulalia Catamo, Stephen J Chanock, Georgia Chenevix-Trench, Marina Ciullo, Tanguy Corre, Fergus J Couch, Angela Cox, Laura Crisponi, Simon S Cross, Francesco Cucca, Kamila Czene, George Davey Smith, Eco J C N de Geus, Renée de Mutsert, Immaculata De Vivo, Ellen W Demerath, Joe Dennis, Alison M Dunning, Miriam Dwek, Mikael Eriksson, Tõnu Esko, Peter A Fasching, Jessica D Faul, Luigi Ferrucci, Nora Franceschini, Timothy M Frayling, Manuela Gago-Dominguez, Massimo Mezzavilla, Montserrat García-Closas, Christian Gieger, Graham G Giles, Harald Grallert, Daniel F Gudbjartsson, Vilmundur Gudnason, Pascal Guénel, Christopher A Haiman, Niclas Håkansson, Per Hall, Caroline Hayward, Chunyan He, Wei He, Gerardo Heiss, Miya K Høffding, John L Hopper, Jouke J Hottenga, Frank Hu, David Hunter, Mohammad A Ikram, Rebecca D Jackson, Micaella D R Joaquim, Esther M John, Peter K Joshi, David Karasik, Sharon L R Kardia, Christiana Kartsonaki, Robert Karlsson, Cari M Kitahara, Ivana Kolcic, Charles Kooperberg, Peter Kraft, Allison W Kurian, Zoltan Kutalik, Martina La Bianca, Genevieve LaChance, Claudia Langenberg, Lenore J Launer, Joop S E Laven, Deborah A Lawlor, Loic Le Marchand, Jingmei Li, Annika Lindblom, Sara Lindstrom, Tricia Lindstrom, Martha Linet, YongMei Liu, Simin Liu, Jian'an Luan, Reedik Mägi, Patrik K E Magnusson, Massimo Mangino, Arto Mannermaa, Brumat Marco, Jonathan Marten, Nicholas G Martin, Hamdi Mbarek, Barbara McKnight, Sarah E Medland, Christa Meisinger, Thomas Meitinger, Cristina Menni, Andres Metspalu, Lili Milani, Roger L Milne, Grant W Montgomery, Dennis O Mook-Kanamori, Antonella Mulas, Anna M Mulligan, Alison Murray, Mike A Nalls, Anne Newman, Raymond Noordam, Teresa Nutile, Dale R Nyholt, Andrew F Olshan, Håkan Olsson, Jodie N Painter, Alpa V Patel, Nancy L Pedersen, Natalia Perjakova, Annette Peters, Ulrike Peters, Paul D P Pharoah, Ozren Polasek, Eleonora Porcu, Bruce M Psaty, Iffat Rahman, Gad Rennert, Hedy S Rennert, Paul M Ridker, Susan M Ring, Antonietta Robino, Lynda M Rose, Frits R Rosendaal, Jacques Rossouw, Igor Rudan, Rico Rueedi, Daniela Ruggiero, Cinzia F Sala, Emmanouil Saloustros, Dale P Sandler, Serena Sanna, Elinor J Sawyer, Chloé Sarnowski, David Schlessinger, Marjanka K Schmidt, Minouk J Schoemaker, Katharina E Schraut, Christopher Scott, Saleh Shekari, Amruta Shrikhande, Albert V Smith, Blair H Smith, Jennifer A Smith, Rossella Sorice, Melissa C Southey, Tim D Spector, John J Spinelli, Meir Stampfer, Doris Stöckl, Joyce B J van Meurs, Konstantin Strauch, Unnur Styrkarsdottir, Anthony J Swerdlow, Toshiko Tanaka, Lauren R Teras, Alexander Teumer, Unnur Þorsteinsdottir, Nicholas J Timpson, Daniela Toniolo, Michela Traglia, Melissa A Troester, Thérèse Truong, Jessica Tyrrell, André G Uitterlinden, Sheila Ulivi, Celine M Vachon, Veronique Vitart, Uwe Völker, Peter Vollenweider, Henry Völzke, Qin Wang, Nicholas J Wareham, Clarice R Weinberg, David R Weir, Amber N Wilcox, Ko Willems van Dijk, Gonneke Willemsen, James F Wilson, Bruce H R Wolffenbuttel, Alicja Wolk, Andrew R Wood, Wei Zhao, Marek Zygmunt, , , , , , , , , Zhengming Chen, Liming Li, Lude Franke, Stephen Burgess, Patrick Deelen, Tune H Pers, Marie Louise Grøndahl, Claus Yding Andersen, Anna Pujol, Andres J Lopez-Contreras, Jeremy A Daniel, Kari Stefansson, Jenny Chang-Claude, Yvonne T van der Schouw, Kathryn L Lunetta, Daniel I Chasman, Douglas F Easton, Jenny A Visser, Susan E Ozanne, Satoshi H Namekawa, Petr Solc, Joanne M Murabito, Ken K Ong, Eva R Hoffmann, Anna Murray, Ignasi Roig, John R B Perry.
      Reproductive longevity is essential for fertility and influences healthy ageing in women1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations3. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.
    DOI:  https://doi.org/10.1038/s41586-021-03779-7
  46. Sci Signal. 2021 Aug 03. pii: eabe0387. [Epub ahead of print]14(694):
    A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation.
    Orna Ernst, Jing Sun, Bin Lin, Balaji Banoth, Michael G Dorrington, Jonathan Liang, Benjamin Schwarz, Kaitlin A Stromberg, Samuel Katz, Sharat J Vayttaden, Clinton J Bradfield, Nadia Slepushkina, Christopher M Rice, Eugen Buehler, Jaspal S Khillan, Daniel W McVicar, Catharine M Bosio, Clare E Bryant, Fayyaz S Sutterwala, Scott E Martin, Madhu Lal-Nag, Iain D C Fraser.
      Noncanonical inflammasome activation by cytosolic lipopolysaccharide (LPS) is a critical component of the host response to Gram-negative bacteria. Cytosolic LPS recognition in macrophages is preceded by a Toll-like receptor (TLR) priming signal required to induce transcription of inflammasome components and facilitate the metabolic reprograming that fuels the inflammatory response. Using a genome-scale arrayed siRNA screen to find inflammasome regulators in mouse macrophages, we identified the mitochondrial enzyme nucleoside diphosphate kinase D (NDPK-D) as a regulator of both noncanonical and canonical inflammasomes. NDPK-D was required for both mitochondrial DNA synthesis and cardiolipin exposure on the mitochondrial surface in response to inflammasome priming signals mediated by TLRs, and macrophages deficient in NDPK-D had multiple defects in LPS-induced inflammasome activation. In addition, NDPK-D was required for the recruitment of TNF receptor-associated factor 6 (TRAF6) to mitochondria, which was critical for reactive oxygen species (ROS) production and the metabolic reprogramming that supported the TLR-induced gene program. NDPK-D knockout mice were protected from LPS-induced shock, consistent with decreased ROS production and attenuated glycolytic commitment during priming. Our findings suggest that, in response to microbial challenge, NDPK-D-dependent TRAF6 mitochondrial recruitment triggers an energetic fitness checkpoint required to engage and maintain the transcriptional program necessary for inflammasome activation.
    DOI:  https://doi.org/10.1126/scisignal.abe0387
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