bims-numges 2020-06-07 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2020‒06‒07
37 papers selected by
Sean Rudd
Karolinska Institutet

Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.
ERK2 Phosphorylates PFAS to Mediate Posttranslational Control of De Novo Purine Synthesis.
HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation.
MTOR signaling orchestrates stress-induced mutagenesis, facilitating adaptive evolution in cancer.
Oncometabolites suppress DNA repair by disrupting local chromatin signalling.
CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage.
POLE Mutation Spectra Are Shaped by the Mutant Allele Identity, Its Abundance, and Mismatch Repair Status.
Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization.
Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression.
m5C modification of mRNA serves a DNA damage code to promote homologous recombination.
Targeting therapeutic vulnerabilities with PARP inhibition and radiation in IDH-mutant gliomas and cholangiocarcinomas.
Design and Synthesis of a Trifunctional Molecular System "Programmed" to Block Epidermal Growth Factor Receptor Tyrosine Kinase, Induce High Levels of DNA Damage, and Inhibit the DNA Repair Enzyme (Poly(ADP-ribose) Polymerase) in Prostate Cancer Cells.
Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks.
Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair.
The MRE11 complex: A versatile toolkit for the repair of broken DNA.
Pharmacological methods to transcriptionally modulate double-strand break DNA repair.
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways.
PARP goes the weasel! Emerging role of PARP inhibitors in acute leukemias.
CDK-Independent and PCNA-Dependent Functions of p21 in DNA Replication.
Genomic Instability in Multiple Myeloma.
HRness in Breast and Ovarian Cancers.
Mechanisms of eukaryotic replisome disassembly.
Damage Incorporated: Discovery of the Potent, Highly Selective, Orally Available ATR Inhibitor BAY 1895344 with Favorable Pharmacokinetic Properties and Promising Efficacy in Monotherapy and in Combination Treatments in Preclinical Tumor Models.
The DNA Damage Response in Telophase: Challenging Dogmas.
The Mre11-Rad50-Nbs1 complex mediates the robust recruitment of Polo to DNA lesions during mitosis.
Defective NADPH production in mitochondrial disease complex I causes inflammation and cell death.
Non-canonical interaction of DNA repair proteins with intact and cleaved AP sites.
The deubiquitinating enzyme UCHL1 promotes resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase.
Rad54 Drives ATP Hydrolysis-Dependent DNA Sequence Alignment during Homologous Recombination.
REV7 is required for processing AID initiated DNA lesions in activated B cells.
The structure of human CST reveals a decameric assembly bound to telomeric DNA.
RBMX is required for activation of ATR on repetitive DNAs to maintain genome stability.
Mechanisms of nucleotide selection by telomerase.
Drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway.
Widely targeted metabolomic analyses unveil the metabolic variations after stable knock-down of NME4 in esophageal squamous cell carcinoma cells.
Design, synthesis and biological evaluation of novel pyrrolo[2,3-d]pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier.
Inosine 5'-diphosphate, a molecular decoy rescues Nucleoside diphosphate kinase from c-MYC G-Quadruplex unfolding.

EMBO J. 2020 Jun 02. e103838

Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.

Joffrey Pelletier, Ferran Riaño-Canalias, Eugènia Almacellas, Caroline Mauvezin, Sara Samino, Sonia Feu, Sandra Menoyo, Ana Domostegui, Marta Garcia-Cajide, Ramon Salazar, Constanza Cortés, Ricard Marcos, Albert Tauler, Oscar Yanes, Neus Agell, Sara C Kozma, Antonio Gentilella, George Thomas.

  Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.

Keywords:   IRBC ; IMPDH; nucleotides; p21; p53

DOI:  https://doi.org/10.15252/embj.2019103838
Mol Cell. 2020 May 28. pii: S1097-2765(20)30302-6. [Epub ahead of print]

ERK2 Phosphorylates PFAS to Mediate Posttranslational Control of De Novo Purine Synthesis.

Eunus S Ali, Umakant Sahu, Elodie Villa, Brendan P O'Hara, Peng Gao, Cynthia Beaudet, Antony W Wood, John M Asara, Issam Ben-Sahra.

  The RAS-ERK/MAPK (RAS-extracellular signal-regulated kinase/mitogen-activated protein kinase) pathway integrates growth-promoting signals to stimulate cell growth and proliferation, at least in part, through alterations in metabolic gene expression. However, examples of direct and rapid regulation of the metabolic pathways by the RAS-ERK pathway remain elusive. We find that physiological and oncogenic ERK signaling activation leads to acute metabolic flux stimulation through the de novo purine synthesis pathway, thereby increasing building block availability for RNA and DNA synthesis, which is required for cell growth and proliferation. We demonstrate that ERK2, but not ERK1, phosphorylates the purine synthesis enzyme PFAS (phosphoribosylformylglycinamidine synthase) at T619 in cells to stimulate de novo purine synthesis. The expression of nonphosphorylatable PFAS (T619A) decreases purine synthesis, RAS-dependent cancer cell-colony formation, and tumor growth. Thus, ERK2-mediated PFAS phosphorylation facilitates the increase in nucleic acid synthesis required for anabolic cell growth and proliferation.

Keywords:  ERK; FGAM; MAPK; PFAS; RAS; cancer; nucleotide synthesis; posttranslational modification; purine metabolism; tumor growth

DOI:  https://doi.org/10.1016/j.molcel.2020.05.001
Cell Rep. 2020 Jun 02. pii: S2211-1247(20)30675-6. [Epub ahead of print]31(9): 107705

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation.

Kavi P M Mehta, Courtney A Lovejoy, Runxiang Zhao, Darren R Heintzman, David Cortez.

  5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases.

Keywords:  APOBEC; DNA damage; DNA repair; HMCES; abasic site; damage tolerance; replication stress; translesion synthesis

DOI:  https://doi.org/10.1016/j.celrep.2020.107705
Science. 2020 Jun 05. 368(6495): 1127-1131

MTOR signaling orchestrates stress-induced mutagenesis, facilitating adaptive evolution in cancer.

Arcadi Cipponi, David L Goode, Justin Bedo, Mark J McCabe, Marina Pajic, David R Croucher, Alvaro Gonzalez Rajal, Simon R Junankar, Darren N Saunders, Pavel Lobachevsky, Anthony T Papenfuss, Danielle Nessem, Max Nobis, Sean C Warren, Paul Timpson, Mark Cowley, Ana C Vargas, Min R Qiu, Daniele G Generali, Shivakumar Keerthikumar, Uyen Nguyen, Niall M Corcoran, Georgina V Long, Jean-Yves Blay, David M Thomas.

In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.

DOI: https://doi.org/10.1126/science.aau8768
Nature. 2020 Jun 03.

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Parker L Sulkowski, Sebastian Oeck, Jonathan Dow, Nicholas G Economos, Lily Mirfakhraie, Yanfeng Liu, Katelyn Noronha, Xun Bao, Jing Li, Brian M Shuch, Megan C King, Ranjit S Bindra, Peter M Glazer.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

DOI: https://doi.org/10.1038/s41586-020-2363-0
EMBO Rep. 2020 Jun 04. e48920

CDC7 kinase promotes MRE11 fork processing, modulating fork speed and chromosomal breakage.

Michael D Rainey, Aisling Quinlan, Chiara Cazzaniga, Sofija Mijic, Oliviano Martella, Jana Krietsch, Anja Göder, Massimo Lopes, Corrado Santocanale.

  The CDC7 kinase is essential for the activation of DNA replication origins and has been implicated in the replication stress response. Using a highly specific chemical inhibitor and a chemical genetic approach, we now show that CDC7 activity is required to coordinate multiple MRE11-dependent processes occurring at replication forks, independently from its role in origin firing. CDC7 localizes at replication forks and, similarly to MRE11, mediates active slowing of fork progression upon mild topoisomerase inhibition. Both proteins are also retained on stalled forks, where they promote fork processing and restart. Moreover, MRE11 phosphorylation and localization at replication factories are progressively lost upon CDC7 inhibition. Finally, CDC7 activity at reversed forks is required for their pathological MRE11-dependent degradation in BRCA2-deficient cells. Thus, upon replication interference CDC7 is a key regulator of fork progression, processing and integrity. These results highlight a dual role for CDC7 in replication, modulating both initiation and elongation steps of DNA synthesis, and identify a key intervention point for anticancer therapies exploiting replication interference.

Keywords:  DNA replication; fork protection; genome stability; kinase inhibitor

DOI:  https://doi.org/10.15252/embr.201948920
Mol Cell. 2020 May 27. pii: S1097-2765(20)30313-0. [Epub ahead of print]

POLE Mutation Spectra Are Shaped by the Mutant Allele Identity, Its Abundance, and Mismatch Repair Status.

Karl P Hodel, Meijuan J S Sun, Nathan Ungerleider, Vivian S Park, Leonard G Williams, David L Bauer, Victoria E Immethun, Jieqiong Wang, Zucai Suo, Hua Lu, James B McLachlan, Zachary F Pursell.

  Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.

Keywords:  DNA polymerase; DNA repair; DNA replication; genomic instability; mismatch repair; mutagenesis

DOI:  https://doi.org/10.1016/j.molcel.2020.05.012
J Biol Chem. 2020 Jun 02. pii: jbc.RA120.014465. [Epub ahead of print]

Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization.

Andrew W Caliri, Stella Tommasi, Steven E Bates, Ahmad Besaratinia.

  To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from lambda phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells, which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors due to oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of oxidative stress-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells, provide insights into the underlying mechanisms of senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.

Keywords:  8-Oxoguanine (8-oxoG); DNA damage; DNA mismatch repair; cancer; immortalization; mouse embryonic fibroblasts (MEF); mutagenesis; oxidative stress; photodynamic therapy; reactive oxygen species; reactive oxygen species (ROS); senescence

DOI:  https://doi.org/10.1074/jbc.RA120.014465
J Clin Invest. 2020 Jun 01. pii: 132876. [Epub ahead of print]130(6): 3253-3269

Parkin ubiquitinates phosphoglycerate dehydrogenase to suppress serine synthesis and tumor progression.

Juan Liu, Cen Zhang, Hao Wu, Xiao-Xin Sun, Yanchen Li, Shan Huang, Xuetian Yue, Shou-En Lu, Zhiyuan Shen, Xiaoyang Su, Eileen White, Bruce G Haffty, Wenwei Hu, Zhaohui Feng.

  Phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme of serine synthesis, is frequently overexpressed in human cancer. PHGDH overexpression activates serine synthesis to promote cancer progression. Currently, PHGDH regulation in normal cells and cancer is not well understood. Parkin, an E3 ubiquitin ligase involved in Parkinson's disease, is a tumor suppressor. Parkin expression is frequently downregulated in many types of cancer, and its tumor-suppressive mechanism is poorly defined. Here, we show that PHGDH is a substrate for Parkin-mediated ubiquitination and degradation. Parkin interacted with PHGDH and ubiquitinated PHGDH at lysine 330, leading to PHGDH degradation to suppress serine synthesis. Parkin deficiency in cancer cells stabilized PHGDH and activated serine synthesis to promote cell proliferation and tumorigenesis, which was largely abolished by targeting PHGDH with RNA interference, CRISPR/Cas9 KO, or small-molecule PHGDH inhibitors. Furthermore, Parkin expression was inversely correlated with PHGDH expression in human breast cancer and lung cancer. Our results revealed PHGDH ubiquitination by Parkin as a crucial mechanism for PHGDH regulation that contributes to the tumor-suppressive function of Parkin and identified Parkin downregulation as a critical mechanism underlying PHGDH overexpression in cancer.

Keywords:  Metabolism; Oncology; Tumor suppressors; Ubiquitin-proteosome system

DOI:  https://doi.org/10.1172/JCI132876
Nat Commun. 2020 Jun 05. 11(1): 2834

m5C modification of mRNA serves a DNA damage code to promote homologous recombination.

Hao Chen, Haibo Yang, Xiaolan Zhu, Tribhuwan Yadav, Jian Ouyang, Samuel S Truesdell, Jun Tan, Yumin Wang, Meihan Duan, Leizhen Wei, Lee Zou, Arthur S Levine, Shobha Vasudevan, Li Lan.

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.

DOI: https://doi.org/10.1038/s41467-020-16722-7
Sci Adv. 2020 Apr;6(17): eaaz3221

Targeting therapeutic vulnerabilities with PARP inhibition and radiation in IDH-mutant gliomas and cholangiocarcinomas.

Yuxiang Wang, Aaron T Wild, Sevin Turcan, Wei H Wu, Carlie Sigel, David S Klimstra, Xiaoxiao Ma, Yongxing Gong, Eric C Holland, Jason T Huse, Timothy A Chan.

Mutations in isocitrate dehydrogenase (IDH) genes occur in multiple cancer types, lead to global changes in the epigenome, and drive tumorigenesis. Yet, effective strategies targeting solid tumors harboring IDH mutations remain elusive. Here, we demonstrate that IDH-mutant gliomas and cholangiocarcinomas display elevated DNA damage. Using multiple in vitro and preclinical animal models of glioma and cholangiocarcinoma, we developed treatment strategies that use a synthetic lethality approach targeting the reduced DNA damage repair conferred by mutant IDH using poly(adenosine 5'-diphosphate) ribose polymerase inhibitors (PARPis). The therapeutic effects are markedly enhanced by cotreatment with concurrent, localized radiation therapy. PARPi-buttressed multimodality therapies may represent a readily applicable approach that is selective for IDH-mutant tumor cells and has potential to improve outcomes in multiple cancers.

DOI: https://doi.org/10.1126/sciadv.aaz3221
J Med Chem. 2020 Jun 02.

Design and Synthesis of a Trifunctional Molecular System "Programmed" to Block Epidermal Growth Factor Receptor Tyrosine Kinase, Induce High Levels of DNA Damage, and Inhibit the DNA Repair Enzyme (Poly(ADP-ribose) Polymerase) in Prostate Cancer Cells.

Julie Schmitt, Shanlong Huang, Elliot Goodfellow, Christopher Williams, Bertrand J Jean-Claude.

Resistance to chemotherapy in advanced cancers can be mediated by different factors such as epidermal growth factor receptor (EGFR) overexpression and DNA repair enzymes. Therefore, current standards of care usually involve combinations of multiple treatments. Here, to reduce the adverse effects of multiple drug combinations and improve outcome, we proposed a single drug approach to block multiple overlapping effects that characterize chemoresistance. Thus, we designed a new linker that allows assembly of multiple functions (e.g., inhibition of EGFR phosphorylation, induction of DNA lesions, and blockade of their repair) into a single molecule. This led to the successful synthesis of a novel and potent combi-molecule JS230. Here, we demonstrated that in resistant prostate cancer cells overexpressing EGFR, it was capable of (a) inhibiting EGFR in a dose-dependent manner, (b) damaging DNA, and (c) sustaining the damage by inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP). The triple mechanism of action of JS230 cumulated into growth inhibitory potency superior to that of classical two- or three-drug combinations.

DOI: https://doi.org/10.1021/acs.jmedchem.9b02008
Nucleic Acids Res. 2020 Jun 05. pii: gkaa483. [Epub ahead of print]

Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks.

Karol Szlachta, Arkadi Manukyan, Heather M Raimer, Sandeep Singh, Anita Salamon, Wenying Guo, Kirill S Lobachev, Yuh-Hwa Wang.

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.

DOI: https://doi.org/10.1093/nar/gkaa483
Nucleic Acids Res. 2020 Jun 06. pii: gkaa489. [Epub ahead of print]

Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair.

Ilona Kalasova, Richard Hailstone, Janin Bublitz, Jovel Bogantes, Winfried Hofmann, Alejandro Leal, Hana Hanzlikova, Keith W Caldecott.

Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.

DOI: https://doi.org/10.1093/nar/gkaa489
DNA Repair (Amst). 2020 May 15. pii: S1568-7864(20)30117-8. [Epub ahead of print]91-92 102869

The MRE11 complex: A versatile toolkit for the repair of broken DNA.

Giordano Reginato, Petr Cejka.

  When DNA breaks, the ends need to be stabilized and processed to facilitate subsequent repair, which can occur by either direct but error-prone end-joining with another broken DNA molecule or a more accurate homology-directed repair by the recombination machinery. At the same time, the presence of broken DNA triggers a signaling cascade that regulates the repair events and cellular progression through the cell cycle. The MRE11 nuclease, together with RAD50 and NBS1 forms a complex termed MRN that participates in all these processes. Although MRE11 was first identified more than 20 years ago, deep insights into its mechanism of action and regulation are much more recent. Here we review how MRE11 functions within MRN, and how the complex is further regulated by CtIP and its phosphorylation in a cell cycle dependent manner. We describe how RAD50, NBS1 and CtIP convert MRE11, exhibiting per se a 3'→5' exonuclease activity, into an ensemble that instead degrades primarily the 5'-terminated strand by endonucleolytic cleavage at DNA break sites to generate 3' overhangs, as required for the initiation of homologous recombination. The unique mechanism of DNA end resection by MRN-CtIP makes it a very flexible toolkit to process DNA breaks with a variety of secondary structures and protein blocks. Such a block can also be the Ku heterodimer, and emerging evidence suggests that MRN-CtIP may often need to remove Ku from DNA ends before initiating homologous recombination. Misregulation of DNA break repair results in mutations and chromosome rearrangements that can drive cancer development. Therefore, a detailed understanding of the underlying processes is highly relevant for human health.

Keywords:  Cancer; Checkpoint; DNA damage and repair; DNA end resection; End-joining; Homologous recombination

DOI:  https://doi.org/10.1016/j.dnarep.2020.102869
Int Rev Cell Mol Biol. 2020 ;pii: S1937-6448(19)30105-4. [Epub ahead of print]354 187-213

Pharmacological methods to transcriptionally modulate double-strand break DNA repair.

Alanna R Kaplan, Peter M Glazer.

  There is much interest in targeting DNA repair pathways for use in cancer therapy, as the effectiveness of many therapeutic agents relies on their ability to cause damage to DNA, and deficiencies in DSB repair pathways can make cells more sensitive to specific cancer therapies. For example, defects in the double-strand break (DSB) pathways, non-homologous end joining (NHEJ) and homology-directed repair (HDR), induce sensitivity to radiation therapy and poly(ADP)-ribose polymerase (PARP) inhibitors, respectively. However, traditional approaches to inhibit DNA repair through small molecule inhibitors have often been limited by toxicity and poor bioavailability. This review identifies several pharmacologic manipulations that modulate DSB repair by reducing expression of DNA repair factors. A number of pathways have been identified that modulate activity of NHEJ and HDR through this mechanism, including growth and hormonal receptor signaling pathways as well as epigenetic modifiers. We also discuss the effects of anti-angiogenic therapy on DSB repair. Preclinically, these pharmacological manipulations of DNA repair factor expression have been shown to increase sensitivity to specific cancer therapies, including ionizing radiation and PARP inhibitors. When applicable, relevant clinical trials are discussed and areas for future study are identified.

Keywords:  Double-strand break DNA repair; Homology-directed repair; Non-homologous end joining; PARP inhibitors; Radiation therapy; Synthetic lethality

DOI:  https://doi.org/10.1016/bs.ircmb.2019.11.003
EMBO J. 2020 Jun 02. e104036

ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways.

Xu Feng, Anthony Tubbs, Chunchao Zhang, Mengfan Tang, Sriram Sridharan, Chao Wang, Dadi Jiang, Dan Su, Huimin Zhang, Zhen Chen, Litong Nie, Yun Xiong, Min Huang, André Nussenzweig, Junjie Chen.

  Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.

Keywords:  ATR; MAVS; cGAS/STING; radiation; type I interferon

DOI:  https://doi.org/10.15252/embj.2019104036
Blood Rev. 2020 May 07. pii: S0268-960X(20)30046-1. [Epub ahead of print] 100696

PARP goes the weasel! Emerging role of PARP inhibitors in acute leukemias.

Claire Fritz, Scott M Portwood, Amanda Przespolewski, Eunice S Wang.

  Poly (ADP-ribose) polymerase (PARP) inhibitors, which induce synthetic lethality of BRCA mutant breast and ovarian cancers, are now under active exploration for treatment of acute leukemias, specifically acute myeloid leukemia (AML). Experimental data has revealed that DNA repair deficiencies similar to those found in BRCA mutant solid tumors function in malignant hematopoietic cells to enhance cell survival and promote therapy resistance. Preclinical studies have demonstrated that inhibition of PARP with a variety of agents can dramatically enhance the efficacy of other therapeutic approaches including cytotoxic and epigenetic chemotherapy, small molecule inhibitors (IDH and FLT3 inhibitors) and antibody drug conjugates. This has led to early stage clinical trials of multiple PARP inhibitors (PARPi) for AML patients. Despite small patient numbers, evidence of modest clinical efficacy and tolerability in combinatorial regimens support the further development of PARP inhibition as a novel therapeutic strategy for AML, particularly in select molecular subsets (MLL rearranged, FLT3 and IDH1 mutant disease.

Keywords:  Acute myeloid leukemia; DNA damage; DNA repair; FLT3 mutation; IDH mutation; Olaparib; PARP inhibition; Talazoparib

DOI:  https://doi.org/10.1016/j.blre.2020.100696
Genes (Basel). 2020 May 28. pii: E593. [Epub ahead of print]11(6):

CDK-Independent and PCNA-Dependent Functions of p21 in DNA Replication.

Sabrina Florencia Mansilla, María Belén de la Vega, Nicolás Luis Calzetta, Sebastián Omar Siri, Vanesa Gottifredi.

  p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.

Keywords:  CDK; DNA replication; PCNA; S phase; TLS; p21CDKN1A

DOI:  https://doi.org/10.3390/genes11060593
Trends Cancer. 2020 May 30. pii: S2405-8033(20)30161-8. [Epub ahead of print]

Genomic Instability in Multiple Myeloma.

David A Alagpulinsa, Raphael E Szalat, Mark C Poznansky, Robert J Shmookler Reis.

  Genomic instability (GIN), an increased tendency to acquire genomic alterations, is a cancer hallmark. However, its frequency, underlying causes, and disease relevance vary across different cancers. Multiple myeloma (MM), a plasma cell malignancy, evolves through premalignant phases characterized by genomic abnormalities. Next-generation sequencing (NGS) methods are deconstructing the genomic landscape of MM across the continuum of its development, inextricably linking malignant transformation and disease progression with increasing acquisition of genomic alterations, and illuminating the mechanisms that generate these alterations. Although GIN drives disease evolution, it also creates vulnerabilities such as dependencies on 'superfluous' repair mechanisms and the induction of tumor-specific antigens that can be targeted. We review the mechanisms of GIN in MM, the associated vulnerabilities, and therapeutic targeting strategies.

Keywords:  DNA damage; DNA repair; genomic instability; immunotherapy; multiple myeloma; replication stress

DOI:  https://doi.org/10.1016/j.trecan.2020.05.006
Int J Mol Sci. 2020 May 28. pii: E3850. [Epub ahead of print]21(11):

HRness in Breast and Ovarian Cancers.

Elizabeth Santana Dos Santos, François Lallemand, Ambre Petitalot, Sandrine M Caputo, Etienne Rouleau.

  Ovarian and breast cancers are currently defined by the main pathways involved in the tumorigenesis. The majority are carcinomas, originating from epithelial cells that are in constant division and subjected to cyclical variations of the estrogen stimulus during the female hormonal cycle, therefore being vulnerable to DNA damage. A portion of breast and ovarian carcinomas arises in the context of DNA repair defects, in which genetic instability is the backdrop for cancer initiation and progression. For these tumors, DNA repair deficiency is now increasingly recognized as a target for therapeutics. In hereditary breast/ovarian cancers (HBOC), tumors with BRCA1/2 mutations present an impairment of DNA repair by homologous recombination (HR). For many years, BRCA1/2 mutations were only screened on germline DNA, but now they are also searched at the tumor level to personalize treatment. The reason of the inactivation of this pathway remains uncertain for most cases, even in the presence of a HR-deficient signature. Evidence indicates that identifying the mechanism of HR inactivation should improve both genetic counseling and therapeutic response, since they can be useful as new biomarkers of response.

Keywords:  BRCA1; BRCA2; DNA repair; breast cancer tumorigenesis; hereditary breast cancer; hereditary ovarian cancer; homologous recombination deficiency; ovarian cancer tumorigenesis

DOI:  https://doi.org/10.3390/ijms21113850
Biochem Soc Trans. 2020 Jun 03. pii: BST20190363. [Epub ahead of print]

Mechanisms of eukaryotic replisome disassembly.

Sara Priego Moreno, Agnieszka Gambus.

  DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

Keywords:  Cullin2-LRR1; TRAIP; genome integrity; p97; replisome disassembly; ubiquitylation

DOI:  https://doi.org/10.1042/BST20190363
J Med Chem. 2020 Jun 05.

Damage Incorporated: Discovery of the Potent, Highly Selective, Orally Available ATR Inhibitor BAY 1895344 with Favorable Pharmacokinetic Properties and Promising Efficacy in Monotherapy and in Combination Treatments in Preclinical Tumor Models.

Ulrich Luecking, Lars Wortmann, Antje Margret Wengner, Julien Lefranc, Philip Lienau, Hans Briem, Gerhard Siemeister, Ulf Boemer, Karsten Denner, Martina Schaefer, Marcus Koppitz, Knut Eis, Florian Bartels, Benjamin Bader, Wilhelm Bone, Dieter Moosmayer, Simon J Holton, Uwe Eberspächer, Joanna Grudzinska-Goebel, Christoph Schatz, Gesa Deeg, Dominik Mumberg, Franz von Nussbaum.

The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Since DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).

DOI: https://doi.org/10.1021/acs.jmedchem.0c00369
Bioessays. 2020 Jun 02. e2000085

The DNA Damage Response in Telophase: Challenging Dogmas.

Matthew K Summers.



Keywords:  DNA damage response; DNA repair; cell cycle; genome stability; mitosis

DOI:  https://doi.org/10.1002/bies.202000085
J Cell Sci. 2020 Jun 02. pii: jcs.244442. [Epub ahead of print]

The Mre11-Rad50-Nbs1 complex mediates the robust recruitment of Polo to DNA lesions during mitosis.

Cedric Landmann, Priscillia Pierre-Elies, Damien Goutte-Gattat, Emilie Montembault, Marie-Charlotte Claverie, Anne Royou.

  The DNA damage sensor, Mre11-Rad50-Nbs1 complex, and Polo kinase are recruited to DNA lesions during mitosis. However, their mechanism of recruitment is elusive. Here, using live-cell imaging combined with the micro-irradiation of single chromosomes, we analyze the dynamics of Polo and Mre11 at DNA lesions during mitosis. The two proteins display distinct kinetics. While Polo kinetics at DSBs are Cdk1-driven, Mre11 promptly but briefly associates with DSBs regardless of the phase of mitosis and re-associates with DSBs in the proceeding interphase. Mechanistically, Polo kinase activity is required for its own recruitment and that of the mitotic proteins BubR1 and Bub3 to DSBs. Moreover, depletion of Rad50 severely impaired Polo kinetics at mitotic DSBs. Conversely, ectopic tethering of Mre11 to chromatin is sufficient to recruit Polo. Our study highlights a novel pathway that links the DSB sensor MRN complex and Polo kinase to initiate a prompt, decisive response to the presence of DNA damage during mitosis.

Keywords:  ACP/C; Anaphase; Bub3; BubR1; Cdc20; Checkpoint; Chromosomes; DNA damage; DNA double strand breaks; Drosophila; Mitosis; Mre11; Polo; Rad50

DOI:  https://doi.org/10.1242/jcs.244442
Nat Commun. 2020 Jun 01. 11(1): 2714

Defective NADPH production in mitochondrial disease complex I causes inflammation and cell death.

Eduardo Balsa, Elizabeth A Perry, Christopher F Bennett, Mark Jedrychowski, Steven P Gygi, John G Doench, Pere Puigserver.

Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions. This bioenergetic failure is thought to underlie pathologies associated with mitochondrial diseases. However, the precise metabolic processes resulting from a defective mitochondrial ETC that compromise cell viability under stress conditions are not entirely understood. We design a whole genome gain-of-function CRISPR activation screen using human mitochondrial disease complex I (CI) mutant cells to identify genes whose increased function rescue glucose restriction-induced cell death. The top hit of the screen is the cytosolic Malic Enzyme (ME1), that is sufficient to enable survival and proliferation of CI mutant cells under nutrient stress conditions. Unexpectedly, this metabolic rescue is independent of increased ATP synthesis through glycolysis or oxidative phosphorylation, but dependent on ME1-produced NADPH and glutathione (GSH). Survival upon nutrient stress or pentose phosphate pathway (PPP) inhibition depends on compensatory NADPH production through the mitochondrial one-carbon metabolism that is severely compromised in CI mutant cells. Importantly, this defective CI-dependent decrease in mitochondrial NADPH production pathway or genetic ablation of SHMT2 causes strong increases in inflammatory cytokine signatures associated with redox dependent induction of ASK1 and activation of stress kinases p38 and JNK. These studies find that a major defect of CI deficiencies is decreased mitochondrial one-carbon NADPH production that is associated with increased inflammation and cell death.

DOI: https://doi.org/10.1038/s41467-020-16423-1
DNA Repair (Amst). 2020 May 05. pii: S1568-7864(20)30092-6. [Epub ahead of print]90 102847

Non-canonical interaction of DNA repair proteins with intact and cleaved AP sites.

Svetlana Khodyreva, Olga Lavrik.

  Apurinic/apyrimidinic (AP) sites are widespread lesions in genomic DNA, arising from a number of exogenous and endogenous sources. These DNA lesions are highly mutagenic and demand efficient repair. The review is devoted to data on searching for previously unrecognized proteins capable of interaction with intact or cleaved AP sites. We mainly focused on proteins that form Schiff base upon this interaction. It is important to note that the aldehyde at the deoxyribose C1 atom both in intact and cleaved AP sites can readily react with nucleophiles of proteins. In most cases, these interactions results in processing of AP sites although the process is less efficient as compared to classical AP/dRP lyases. The biological role of these interactions in providing of backup pathways of DNA repair processes is discussed.

Keywords:  ABH1; AP site; Ape1; Base excision repair; DNA-protein cross-link; Deoxyribose phosphate; GAPDH; HMGA; HMGB1; Ku antigen; PARP

DOI:  https://doi.org/10.1016/j.dnarep.2020.102847
Theranostics. 2020 ;10(13): 6048-6060

The deubiquitinating enzyme UCHL1 promotes resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase.

Xinyuan Ding, Yuting Gu, Min Jin, Xin Guo, Sudong Xue, Caihong Tan, Jiefang Huang, Wanlin Yang, Mingxing Xue, Qianjun Zhou, Wenjuan Wang, Yanyun Zhang.

  Rationale: Resistance to pemetrexed (PEM)-based chemotherapy is a major cause of progression in non-small cell lung cancer (NSCLC) patients. The deubiquitinating enzyme UCHL1 was recently found to play important roles in chemoresistance and tumor progression. However, the potential roles and mechanisms of UCHL1 in PEM resistance remain unclear. Methods: Bioinformatics analyses and immunohistochemistry were used to evaluate UCHL1 expression in NSCLC specimens. Kaplan-Meier analysis with the log-rank test was used for survival analyses. We established PEM-resistant NSCLC cell lines by exposing them to step-wise increases in PEM concentrations, and in vitro and in vivo assays were used to explore the roles and mechanisms of UCHL1 in PEM resistance using the NSCLC cells. Results: In chemoresistant tumors from NSCLC patients, UCHL1 was highly expressed and elevated UCHL1 expression was strongly associated with poor outcomes. Furthermore, UCHL1 expression was significantly upregulated in PEM-resistant NSCLC cells, while genetic silencing or inhibiting UCHL1 suppressed resistance to PEM and other drugs in NSCLC cells. Mechanistically, UCHL1 promoted PEM resistance in NSCLC by upregulating the expression of thymidylate synthase (TS), based on reduced TS expression after UCHL1 inhibition and re-emergence of PEM resistance upon TS restoration. Furthermore, UCHL1 upregulated TS expression, which mitigated PEM-induced DNA damage and cell cycle arrest in NSCLC cells, and also conferred resistance to PEM and other drugs. Conclusions: It appears that UCHL1 promotes PEM resistance by upregulating TS in NSCLC cells, which mitigated DNA damage and cell cycle arrest. Thus, UCHL1 may be a therapeutic target for overcoming PEM resistance in NSCLC patients.

Keywords:  UCHL1; chemoresistance; non-small cell lung cancer; pemetrexed; thymidylate synthase

DOI:  https://doi.org/10.7150/thno.42096
Cell. 2020 May 21. pii: S0092-8674(20)30562-6. [Epub ahead of print]

Rad54 Drives ATP Hydrolysis-Dependent DNA Sequence Alignment during Homologous Recombination.

J Brooks Crickard, Corentin J Moevus, Youngho Kwon, Patrick Sung, Eric C Greene.

  Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.

Keywords:  DNA curtain; DNA double-strand break; Rad51; Rad54; homologous recombination; homology search; motor protein; replication protein A; single molecule

DOI:  https://doi.org/10.1016/j.cell.2020.04.056
Nat Commun. 2020 Jun 04. 11(1): 2812

REV7 is required for processing AID initiated DNA lesions in activated B cells.

Dingpeng Yang, Ying Sun, Jingjing Chen, Ying Zhang, Shuangshuang Fan, Min Huang, Xia Xie, Yanni Cai, Yafang Shang, Tuantuan Gui, Liming Sun, Jiazhi Hu, Junchao Dong, Leng-Siew Yeap, Xiaoming Wang, Wei Xiao, Fei-Long Meng.

Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. DNA double-strand break response (DSBR) factors promote rearrangement in CSR, while translesion synthesis (TLS) polymerases generate mutations in SHM. REV7, a component of TLS polymerase zeta, is also a downstream effector of 53BP1-RIF1 DSBR pathway. Here, we study the multi-functions of REV7 and find that REV7 is required for the B cell survival upon AID-deamination, which is independent of its roles in DSBR, G2/M transition or REV1-mediated TLS. The cell death in REV7-deficient activated B cells can be fully rescued by AID-deficiency in vivo. We further identify that REV7-depedent TLS across UNG-processed apurinic/apyrimidinic sites is required for cell survival upon AID/APOBEC deamination. This study dissects the multiple roles of Rev7 in antibody diversification, and discovers that TLS is not only required for sequence diversification but also B cell survival upon AID-initiated lesions.

DOI: https://doi.org/10.1038/s41467-020-16632-8
Science. 2020 Jun 05. 368(6495): 1081-1085

The structure of human CST reveals a decameric assembly bound to telomeric DNA.

Ci Ji Lim, Alexandra T Barbour, Arthur J Zaug, Karen J Goodrich, Allison E McKay, Deborah S Wuttke, Thomas R Cech.

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.

DOI: https://doi.org/10.1126/science.aaz9649
Cell Death Differ. 2020 Jun 03.

RBMX is required for activation of ATR on repetitive DNAs to maintain genome stability.

Tian Zheng, Haoxian Zhou, Xiaocui Li, Di Peng, Yiding Yang, Yanru Zeng, Haiying Liu, Jian Ren, Yong Zhao.

ATR is a master regulator of cell response to replication stress. Adequate activation of ATR is essential for preventing genome aberrance induced by replication defect. However, the mechanism underlying ATR activation is not fully understood. Here, we identify that RBMX is an ssDNA binding protein that orchestrates a novel pathway to activate ATR. Using super-resolution STORM, we observe that RBMX and RPA bind to adjacent but nonoverlapping sites on ssDNA in response to replication stress. RBMX then binds to and facilitates positioning of TopBP1, which activates nearby ATR associated with RPA. In addition, ATR activation by ssDNA-RBMX-TopBP1 is independent of ssDNA-dsDNA junction and 9-1-1 complex. ChIP-seq analysis reveals that RBMX/RPA are highly enriched on repetitive DNAs, which are considered as fragile sites with high replication stress. RBMX depletion leads to defective localization of TopBP1 to replication stressed sites and inadequate activation of ATR. Furthermore, cells with deficient RBMX demonstrate replication defect, leading to formation of micronuclei and a high rate of sister-chromatin exchange, indicative of genome instability. Together, the results identify a new ssDNA-RBMX-TopBP1 pathway that is specifically required for activation of ATR on repetitive DNAs. Therefore, RBMX is a key factor to ensure genome stability during replication.

DOI: https://doi.org/10.1038/s41418-020-0570-8
Elife. 2020 Jun 05. pii: e55438. [Epub ahead of print]9

Mechanisms of nucleotide selection by telomerase.

Matthew A Schaich, Samantha L Sanford, Griffin A Welfer, Samuel A Johnson, Thu H Khoang, Patricia L Opresko, Bret D Freudenthal.

  Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.

Keywords:  T. castaneum; biochemistry; chemical biology; fidelity; molecular biophysics; reverse transcriptase; ribonucleotide; structural biology; telomerase

DOI:  https://doi.org/10.7554/eLife.55438
Antiviral Res. 2020 May 30. pii: S0166-3542(20)30237-0. [Epub ahead of print] 104823

Drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway.

Sunrui Chen, Yining Wang, Pengfei Li, Yuebang Yin, Marcel Bijvelds, Hugo de Jonge, Maikel P Peppelenbosch, Denis Kainov, Qiuwei Pan.

Although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. Since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. We identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. We confirmed this effect in 2D cell cultures and 3D cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. Supplementation of UTP or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. Our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients.

DOI: https://doi.org/10.1016/j.antiviral.2020.104823
Mol Cell Biochem. 2020 Jun 05.

Widely targeted metabolomic analyses unveil the metabolic variations after stable knock-down of NME4 in esophageal squamous cell carcinoma cells.

Shutao Zheng, Tao Liu, Qing Liu, Lifei Yang, Qiqi Zhang, Xiujuan Han, Tongxue Shen, Xiao Zhang, Xiaomei Lu.

  NME4, also designated nm23-H4 or NDPK-D, has been known for years for its well-established roles in the synthesis of nucleoside triphosphates, though; little has been known regarding the differential metabolites involved as well as the biological roles NME4 plays in proliferation and invasion of esophageal squamous cell carcinoma (ESCC) cells. To understand the biological roles of NME4 in ESCC cells, lentiviral-based short hairpin RNA interference (shRNA) vectors were constructed and used to stably knock down NME4. Then, the proliferative and invasive variations were assessed using MTT, Colony formation and Transwell assays. To understand the metabolites involved after silencing of NME4 in ESCC cells, widely targeted metabolomic screening was taken. It was discovered that silencing of NME4 can profoundly suppress the proliferation and invasion in ESCC cells in vitro. Metabolically, a total of 11 differential metabolites were screened. KEGG analyses revealed that Tryptophan, Riboflavin, Purine, Nicotinate, lysine degradation, and Linoleic acid metabolism were also involved in addition to the well-established nucleotides metabolism. Some of these differential metabolites, say, 2-Picolinic Acid, Nicotinic Acid and Pipecolinic Acid were suggested to be associated with tumor immunomodulation. The data we described here support the idea that metabolisms occurred in mitochondrial was closely related to tumor immunity.

Keywords:  ESCC; Invasion; Metabolism; NME4; Widely targeted metabolomics

DOI:  https://doi.org/10.1007/s11010-020-03768-w
Bioorg Med Chem. 2020 Jun 15. pii: S0968-0896(20)30374-6. [Epub ahead of print]28(12): 115544

Design, synthesis and biological evaluation of novel pyrrolo[2,3-d]pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier.

Lalit K Golani, Farhana Islam, Carrie O'Connor, Aamod S Dekhne, Zhanjun Hou, Larry H Matherly, Aleem Gangjee.

  Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -β over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRβ-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -β over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -β over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRβ-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.

Keywords:  Folate receptor; Heteroatom bridge; Pyrrolo[2,3-d]pyrimidine; Reduced folate carrier; Selective uptake

DOI:  https://doi.org/10.1016/j.bmc.2020.115544
Biochim Biophys Acta Gen Subj. 2020 May 31. pii: S0304-4165(20)30161-6. [Epub ahead of print] 129649

Inosine 5'-diphosphate, a molecular decoy rescues Nucleoside diphosphate kinase from c-MYC G-Quadruplex unfolding.

Pallabi Sengupta, Subhrangsu Chatterjee.

  BACKGROUND: The transcription-inhibitory G-Quadruplex(Pu27-GQ) atc-MYCpromoter is challenging to target due to structural heterogeneity. Nucleoside diphosphate kinase (NM23-H2) specifically binds and unfolds Pu27-GQ to increasec-MYC transcription. Here, we used Inosine 5'-diphosphate (IDP) to disrupt NM23-H2-Pu27-GQ interactions and arrest c-MYC transcription without compromising NM23-H2-mediated kinase properties.METHODS: Site-directed mutagenesis,31P NMR and STD-NMR studies delineate the epitope of NM23-H2-IDP complex andcharacterize specific amino acids in NM23-H2 involved in Pu27-GQ and IDP interactions. Immunoprecipitations and phosphohistidine-immunoblotsreveal how IDP blocks NM23-H2-Pu27 associationto downregulatec-MYC transcription in MDAMB-231 cellsexempting NM23-H2-mediated kinase properties.
RESULTS: NMR studies show that IDP binds to the Guanosine diphosphate-binding pocket of NM23-H2 (KD = 5.0 ± 0.276 μM). Arg88-drivenhydrogen bondsto the terminal phosphate of IDP restricts P-O-P bond-rotation increasing its pKa (∆pKa = 0.85 ± 0.0025)0.9-inosinyl moiety of IDP is stacked over Phe60 phenyl ring drivingtrans-conformation of inosine and axial geometry of pyrophosphates. Chromatin immunoprecipitationsrevealed that these interactions rescue NM23-H2-driven Pu27-GQ unfolding, which triggers Nucleolin recruitment and lowers Sp1 occupancy at c-MYCpromoterstabilizing Pu27-GQ. This silences c-MYC transcriptionthat reduces c-MYC-Sp1 associationamplifying Sp1 recruitment across P21 promoter stimulating P21 transcription and G2/M arrest.
CONCLUSIONS: IDP synergizes the effects of Pu27-GQ-interacting compounds to abrogate c-MYC transcription and induce apoptosis inMDAMB-231 cells by disruptingNM23-H2-Pu27-GQ interactions without affectingNM23-H2-mediated kinase properties.
GENERAL SIGNIFICANCE: Our study provides a pragmatic approach for developing NM23-H2-targeting regulators to rescue NM23-H2 binding at structurally ambiguous Pu27-GQ that synergizes the anti-tumorigenic effects of GQ-based therapeuticswith minimized off-target effects.

Keywords:  C-MYC; Cell cycle; G-Quadruplex; Inosine diphosphate; NM23-H2; Transcription

DOI:  https://doi.org/10.1016/j.bbagen.2020.129649