bims-numges 2020-09-13 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2020–09–13
35 papers selected by
Sean Rudd, Karolinska Institutet

SLC25A51 is a mammalian mitochondrial NAD+ transporter.
Exploitation of DHODH and p53 activation as therapeutic targets - a case study in polypharmacology.
Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.
INO80C Remodeler Maintains Genomic Stability by Preventing Promiscuous Transcription at Replication Origins.
Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo.
Functions of TopBP1 in preserving genome integrity during mitosis.
ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks.
Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue.
Checkpoint kinase‑1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair.
Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.
Targeting the DNA damage response for patients with lymphoma: Preclinical and clinical evidences.
Autophagy Contributes to the Maintenance of Genomic Integrity by Reducing Oxidative Stress.
Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 induces DNA damage, cell death, and suppression of tumor growth in preclinical models of Ewing sarcoma.
A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age.
The trajectory of intrahelical lesion recognition and extrusion by the human 8-oxoguanine DNA glycosylase.
Protective Effect of Thymidine on DNA Damage Induced by Hydrogen Peroxide in Human Hepatocellular Cancer Cells.
TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks.
DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.
SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.
Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage.
CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage.
The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks.
AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers.
Cancer proteome and metabolite changes linked to SHMT2.
Pif1, RPA and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis.
Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells.
Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch.
Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells.
DNA Methylation-Based Epigenetic Repression of SLC22A4 Promotes Resistance to Cytarabine in Acute Myeloid Leukemia.
Hedgehog signaling enables repair of ribosomal DNA double-strand breaks.
Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.
TOR mediates the autophagy response to altered nucleotide homeostasis in a ribonuclease mutant.
Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents.
New insights into purine metabolism in metabolic diseases: role of xanthine oxidoreductase activity.
Treatment of hairy cell leukemia.

Nature. 2020 Sep 09.

SLC25A51 is a mammalian mitochondrial NAD+ transporter.

Timothy S Luongo, Jared M Eller, Mu-Jie Lu, Marc Niere, Fabio Raith, Caroline Perry, Marc R Bornstein, Paul Oliphint, Lin Wang, Melanie R McReynolds, Marie E Migaud, Joshua D Rabinowitz, F Brad Johnson, Kai Johnsson, Mathias Ziegler, Xiaolu A Cambronne, Joseph A Baur.

Mitochondria require nicotinamide adenine dinucleotide (NAD+) in order to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2 but their very existence is controversial in mammals3-5. Here we demonstrate that mammalian mitochondria are capable of taking up intact NAD+ and identify SLC25A51 (an essential6,7 mitochondrial protein of previously unknown function, also known as MCART1) as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial but not whole-cell NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or a nearly identical paralog, SLC25A52, increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as the first transporter capable of importing NAD+ into mammalian mitochondria.

DOI: https://doi.org/10.1038/s41586-020-2741-7
J Biol Chem. 2020 Sep 08. pii: jbc.RA119.012056. [Epub ahead of print]

Exploitation of DHODH and p53 activation as therapeutic targets - a case study in polypharmacology.

Marcus J G W Ladds, Gergana Popova, Andrés Pastor-Fernández, Srinivasaraghavan Kannan, Ingeborg M M van Leeuwen, Maria Håkansson, Björn Walse, Fredrik Tholander, Ravi Bhatia, Chandra S Verma, David P Lane, Sonia Laín.

  The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, DHODH, and the blockage of uridine transport into cells. These findings hold a three-fold significance; firstly, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; secondly, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.

Keywords:  cell death; mitochondria; molecular modeling; molecular pharmacology; nucleoside/nucleotide biosynthesis; nucleoside/nucleotide transport; p53; tumor cell biology

DOI:  https://doi.org/10.1074/jbc.RA119.012056
Nat Commun. 2020 Sep 10. 11(1): 4534

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.

Lisa Prendergast, Urszula L McClurg, Rossitsa Hristova, Rolando Berlinguer-Palmini, Sarah Greener, Katie Veitch, Inmaculada Hernandez, Philippe Pasero, Daniel Rico, Jonathan M G Higgins, Anastas Gospodinov, Manolis Papamichos-Chronakis.

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.

DOI: https://doi.org/10.1038/s41467-020-18306-x
Cell Rep. 2020 Sep 08. pii: S2211-1247(20)31095-0. [Epub ahead of print]32(10): 108106

INO80C Remodeler Maintains Genomic Stability by Preventing Promiscuous Transcription at Replication Origins.

Salih Topal, Christopher Van, Yong Xue, Michael F Carey, Craig L Peterson.

  The proper coordination of transcription with DNA replication and repair is central for genomic stability. We investigate how the INO80C chromatin remodeling enzyme might coordinate these genomic processes. We find that INO80C co-localizes with the origin recognition complex (ORC) at yeast replication origins and is bound to replication initiation sites in mouse embryonic stem cells (mESCs). In yeast, INO80C recruitment requires origin sequences but does not require ORC, suggesting that recruitment is independent of pre-replication complex assembly. In both yeast and ESCs, INO80C co-localizes at origins with Mot1 and NC2 transcription factors, and genetic studies suggest that they function together to promote genome stability. Interestingly, nascent transcript sequencing demonstrates that INO80C and Mot1 prevent pervasive transcription through origin sequences, and absence of these factors leads to formation of new DNA double-strand breaks. We propose that INO80C and Mot1/NC2 function through distinct pathways to limit origin transcription, maintaining genomic stability.

Keywords:  DSB; INO80; Mot1; NC2; NET-seq; ORC; chromatin; ncRNA; replication

DOI:  https://doi.org/10.1016/j.celrep.2020.108106
Nucleic Acids Res. 2020 Sep 05. pii: gkaa718. [Epub ahead of print]

Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo.

Zhengping Shao, Brian J Lee, Élise Rouleau-Turcotte, Marie-France Langelier, Xiaohui Lin, Verna M Estes, John M Pascal, Shan Zha.

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

DOI: https://doi.org/10.1093/nar/gkaa718
Semin Cell Dev Biol. 2020 Sep 07. pii: S1084-9521(19)30094-1. [Epub ahead of print]

Functions of TopBP1 in preserving genome integrity during mitosis.

Jonas Bagge, Vibe H Oestergaard, Michael Lisby.

  TopBP1/Rad4/Dpb11 is an essential eukaryotic protein with important roles in DNA replication, DNA repair, DNA damage checkpoint activation, and chromosome segregation. TopBP1 serves as a scaffold to assemble protein complexes in a phosphorylation-dependent manner via its multiple BRCT-repeats. Recently, it has become clear that TopBP1 is repurposed to scaffold different processes dependent on cell cycle regulated changes in phosphorylation of client proteins. Here we review the functions of human TopBP1 in maintaining genome integrity during mitosis.

Keywords:  Anaphase Bridges; Homologous recombination; MDC1; MiDAS; Mitosis; SLX4; TopBP1

DOI:  https://doi.org/10.1016/j.semcdb.2020.08.009
Nucleic Acids Res. 2020 Sep 05. pii: gkaa723. [Epub ahead of print]

ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks.

Sébastien Britton, Pauline Chanut, Christine Delteil, Nadia Barboule, Philippe Frit, Patrick Calsou.

Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

DOI: https://doi.org/10.1093/nar/gkaa723
Curr Genet. 2020 Sep 09.

Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue.

Rowin Appanah, David Jones, Benoît Falquet, Ulrich Rass.

  The disease-associated nuclease-helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.

Keywords:  Chromosome stability; DNA replication fork; DNA replication stress; DNA2 nuclease–helicase; Homologous recombination; Seckel syndrome

DOI:  https://doi.org/10.1007/s00294-020-01106-7
Oncol Rep. 2020 Sep 07.

Checkpoint kinase‑1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair.

Zhuoyi Fan, Huacheng Luo, Jie Zhou, Fangce Wang, Wenjun Zhang, Jian Wang, Shuo Li, Qian Lai, Yueshuang Xu, Guangming Wang, Aibin Liang, Jun Xu.

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA‑mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK‑8, and comet assay. The results demonstrated that shRNA‑induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.

DOI: https://doi.org/10.3892/or.2020.7757
Nucleic Acids Res. 2020 Sep 05. pii: gkaa686. [Epub ahead of print]

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.

Andrea Zurita Leal, Marie Schwebs, Emma Briggs, Nadine Weisert, Helena Reis, Leandro Lemgruber, Katarina Luko, Jonathan Wilkes, Falk Butter, Richard McCulloch, Christian J Janzen.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

DOI: https://doi.org/10.1093/nar/gkaa686
Cancer Treat Rev. 2020 Aug 19. pii: S0305-7372(20)30128-6. [Epub ahead of print]90 102090

Targeting the DNA damage response for patients with lymphoma: Preclinical and clinical evidences.

Laura Carrassa, Ilaria Colombo, Giovanna Damia, Francesco Bertoni.

  The DNA damage response (DDR) is a well-coordinated cellular network activated by DNA damage. The unravelling of the key players in DDR, their specific inactivation in different tumor types and the synthesis of specific chemical inhibitors of DDR represent a new hot topic in cancer therapy. In this article, we will review the importance of DDR in lymphoma development and how this can be exploited therapeutically. Specifically, we will focus on CHK1, WEE1, ATR, DNA-PK and PARP inhibitors, for which preclinical data as single agents or in combination has been accumulating, fostering their clinical development. The few available clinical data on these inhibitors will also be discussed.

Keywords:  ATM; ATR; CHK1; DNA damage response; DNA-PK; LYMPHOMA; PARP; WEE1

DOI:  https://doi.org/10.1016/j.ctrv.2020.102090
Oxid Med Cell Longev. 2020 ;2020 2015920

Autophagy Contributes to the Maintenance of Genomic Integrity by Reducing Oxidative Stress.

Wenqing Bu, Xiaohe Hao, Tingting Yang, Jing Wang, Qiao Liu, Xiyu Zhang, Xi Li, Yaoqin Gong, Changshun Shao.

Autophagy has been well documented to play an important role in maintaining genomic stability. However, in addition to directly engulfing and digesting the damaged organelles and chromatin fragments, autophagy can affect many cellular processes including DNA damage response, regulation of redox homeostasis, and cell division; it remains to be determined to what extent each of those processes contributes to the maintenance of genomic stability. We here examined the role of autophagy-dependent redox regulation in the maintenance of genomic stability in two cancer cell lines (HT1080 and U2OS) and mesenchymal stem cells (MSCs) using micronuclei MN, also referred to as cytoplasmic chromatin fragments, as a marker. Our results showed that the spontaneous and genotoxic stress-induced frequencies of MN in cancer cells were significantly reduced by autophagy activators rapamycin and Torin1, and the reduction in MN was accompanied by a reduction in reactive oxygen species (ROS). Increased micronucleation in senescent MSCs, in which autophagic flux is blocked, was also attenuated by rapamycin, together with a reduction in ROS. Inhibition of autophagy by chloroquine (CQ) or ATG5 depletion, on the other hand, resulted in an increased frequency of MN, though a ROS elevation in response to autophagy inhibition was only observed in MSCs. Importantly, the induction of MN by autophagy inhibition in MSCs could be abrogated by antioxidant N-acetylcysteine (NAC). In contrast to the reported impairment of CHK1 activation in Atg7-deficient mouse embryonic fibroblasts, we found that the level of phosphorylated CHK1 was increased by CQ or ATG5 depletion but decreased by rapamycin or Torin1, suggesting that the increased genomic instability by defective autophagy is not caused by insufficient activation of CHK1-homologous recombination cascade. Together, our findings suggest that redox homeostasis regulated by autophagy contributes substantially to the maintenance of genomic stability in certain contexts.

DOI: https://doi.org/10.1155/2020/2015920
Oncogenesis. 2020 Sep 10. 9(9): 80

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 induces DNA damage, cell death, and suppression of tumor growth in preclinical models of Ewing sarcoma.

Anna E Gibson, Choh Yeung, Sameer H Issaq, Victor J Collins, Michael Gouzoulis, Yiping Zhang, Jiuping Ji, Arnulfo Mendoza, Christine M Heske.

NAMPT mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergetics and provides a necessary substrate for functions essential to rapidly proliferating cancer cells. In this study, we evaluated the efficacy and mechanisms of action of OT-82, a novel, high-potency NAMPT inhibitor with a favorable toxicity profile, in preclinical models of Ewing sarcoma (EWS), an aggressive pediatric malignancy with previously reported selective sensitivity to NAMPT inhibition. We show that OT-82 decreased NAD concentration and impaired proliferation of EWS cells in a dose-dependent manner, with IC50 values in the single-digit nanomolar range. Notably, genetic depletion of NAMPT phenocopied pharmacological inhibition. On-target activity of OT-82 was confirmed with the addition of NMN, the product of NAMPT, which rescued NAD concentration and EWS cellular viability. Mechanistically, OT-82 treatment resulted in impaired DNA damage repair through loss of PARP activity, G2 cell-cycle arrest, and apoptosis in EWS cells. Additional consequences of OT-82 treatment included reduction of glycolytic and mitochondrial activity. In vivo, OT-82 impaired tumor growth and prolonged survival in mice bearing EWS xenografts. Importantly, antitumor effect correlated with pharmacodynamic markers of target engagement. Furthermore, combining low-dose OT-82 with low doses of agents augmenting DNA damage demonstrated enhanced antitumor activity in vitro and in vivo. Thus, OT-82 treatment represents a potential novel targeted approach for the clinical treatment of EWS.

DOI: https://doi.org/10.1038/s41389-020-00264-0
Int J Mol Sci. 2020 Sep 09. pii: E6600. [Epub ahead of print]21(18):

A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age.

Nicola Winkelbeiner, Viktoria K Wandt, Franziska Ebert, Kristina Lossow, Ezgi E Bankoglu, Maximilian Martin, Aswin Mangerich, Helga Stopper, Julia Bornhorst, Anna P Kipp, Tanja Schwerdtle.

  Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.

Keywords:  DNA damage; DNA damage response; ageing; base excision repair (incision activity); liver; maintenance of genomic integrity; poly(ADP-ribosyl)ation; sex

DOI:  https://doi.org/10.3390/ijms21186600
Nat Commun. 2020 09 07. 11(1): 4437

The trajectory of intrahelical lesion recognition and extrusion by the human 8-oxoguanine DNA glycosylase.

Uddhav K Shigdel, Victor Ovchinnikov, Seung-Joo Lee, Jenny A Shih, Martin Karplus, Kwangho Nam, Gregory L Verdine.

Efficient search for DNA damage embedded in vast expanses of the DNA genome presents one of the greatest challenges to DNA repair enzymes. We report here crystal structures of human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, that interact with the DNA containing the damaged base oxoG and the normal base G while they are nested in the DNA helical stack. The structures reveal that hOGG1 engages the DNA using different protein-DNA contacts from those observed in the previously determined lesion recognition complex and other hOGG1-DNA complexes. By applying molecular dynamics simulations, we have determined the pathways taken by the lesion and normal bases when extruded from the DNA helix and their associated free energy profiles. These results reveal how the human oxoG DNA glycosylase hOGG1 locates the lesions inside the DNA helix and facilitates their extrusion for repair.

DOI: https://doi.org/10.1038/s41467-020-18290-2
ACS Omega. 2020 Sep 01. 5(34): 21796-21804

Protective Effect of Thymidine on DNA Damage Induced by Hydrogen Peroxide in Human Hepatocellular Cancer Cells.

Yan Li, Jianru Guo, Huixia Zhang, Christopher Wai Kei Lam, Wendi Luo, Hua Zhou, Wei Zhang.

Intracellular ribonucleotide (RN) and deoxyribonucleotide (dRN) pool sizes are critical for the fidelity of DNA synthesis. They are likely to be severely perturbed by many factors which disrupt the integrity and stability of DNA, leading to DNA damage. Exogenously supplied nucleosides are able to increase the deoxynucleoside triphosphate pools, then reverse the DNA damage, and decrease the oncogene-induced transformation dramatically. In this study, the impact of thymidine on the hydrogen peroxide (H2O2)-induced DNA damage was investigated in HepG2 liver cancer cells. From the result of the comet assay, the tail length of cells in the thymidine 600 μM + H2O2 1.0 mM group was dramatically decreased from 42.1 ± 10.8 to 21.9 ± 2.4 μm compared to that exposed with 1.0 mM H2O2 (p < 0.05), suggesting that pretreatment of thymidine reduced the DNA damage of HepG2 cells. Although the RN and dRN contents decreased in the damage group, most of them presented increasing tendency when pretreated with thymidine, especially the key metabolites dCTP, which was mainly related with the decline in the rate of DNA synthesis. The restoration also showed a significant G0/G1 phase arrest of cell cycle progression from 44.6 ± 2.2 to 56.6 ± 0.4% after pretreated with thymidine (p < 0.05). In conclusion, our data demonstrated that the pretreatment with thymidine had a potential protective ability against oxidative damage for DNA in HepG2 cells through the perturbation of RN and dRN pools as well as cell cycle arrest, which should provide new insights into the molecular basis of preventing H2O2-induced oxidative DNA damage in mammalian cells.

DOI: https://doi.org/10.1021/acsomega.0c02843
Proc Natl Acad Sci U S A. 2020 Sep 08. pii: 202004122. [Epub ahead of print]

TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks.

Min Li, Xiaohua Xu, Chou-Wei Chang, Yilun Liu.

  In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription-replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.

Keywords:  DNA replication; PCNA; RECQ5; SUMO2; TRIM28

DOI:  https://doi.org/10.1073/pnas.2004122117
Mutagenesis. 2020 Sep 11. pii: geaa023. [Epub ahead of print]

DNA ligase I variants fail in the ligation of mutagenic repair intermediates with mismatches and oxidative DNA damage.

Qun Tang, Pradnya Kamble, Melike Çağlayan.

DNA ligase I (LIG1) joins DNA strand breaks during DNA replication and repair transactions and contributes to genome integrity. The mutations (P529L, E566K, R641L and R771W) in LIG1 gene are described in patients with LIG1-deficiency syndrome that exhibit immunodeficiency. LIG1 senses 3'-DNA ends with a mismatch or oxidative DNA base inserted by a repair DNA polymerase. However, the ligation efficiency of the LIG1 variants for DNA polymerase-promoted mutagenesis products with 3'-DNA mismatches or 8-oxo-2'-deoxyguanosine (8-oxodG) remains undefined. Here, we report that R641L and R771W fail in the ligation of nicked DNA with 3'-8-oxodG, leading to an accumulation of 5'-AMP-DNA intermediates in vitro. Moreover, we found that the presence of all possible 12 non-canonical base pairs variously impacts the ligation efficiency by P529L and R771W depending on the architecture at the DNA end, whereas E566K exhibits no activity against all substrates tested. Our results contribute to the understanding of the substrate specificity and mismatch discrimination of LIG1 for mutagenic repair intermediates and the effect of non-synonymous mutations on ligase fidelity.

DOI: https://doi.org/10.1093/mutage/geaa023
Nucleic Acids Res. 2020 Sep 05. pii: gkaa754. [Epub ahead of print]

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.

Andreas Mund, Tobias Schubert, Hannah Staege, Sarah Kinkley, Kerstin Reumann, Malte Kriegs, Lauriane Fritsch, Valentine Battisti, Slimane Ait-Si-Ali, Anne-Sophie Hoffbeck, Evi Soutoglou, Hans Will.

DOI: https://doi.org/10.1093/nar/gkaa754
J Biol Chem. 2020 Sep 10. pii: jbc.RA120.014530. [Epub ahead of print]

Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage.

Yu Xu, Akanksha Manghrani, Bei Liu, Honglue Shi, Uyen Pham, Amy Liu, Hashim M Al-Hashimi.

  As the Watson-Crick faces of nucleobases are protected in double-stranded DNA (dsDNA), it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to single-stranded DNA has not been quantified. Watson-Crick base-pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base-pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base-pairs in dsDNA only conferred ~130-fold protection against adenine-N1 methylation and this protection was reduced to ~40-fold for A(syn)-T Hoogsteen base-pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base-pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

Keywords:  DNA damage; DNA dynamics; DNA methylation; DNA repair; DNA sequencing; DNA structure; N1-methylated adenine; alkB; echinomycin

DOI:  https://doi.org/10.1074/jbc.RA120.014530
Cell Death Dis. 2020 Sep 10. 11(9): 740

CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage.

Marie-Christine Albert, Kerstin Brinkmann, Wojciech Pokrzywa, Saskia Diana Günther, Martin Krönke, Thorsten Hoppe, Hamid Kashkar.

The BH3-only protein NOXA is a regulator of mitochondrial apoptosis by specifically antagonizing the anti-apoptotic protein MCL-1. Here we show that the E3 ubiquitin ligase CHIP controls NOXA stability after DNA damage. Our findings reveal that CHIP and MCL-1 are binding partners of NOXA and differentially define the fate of NOXA. Whereas NOXA is initially targeted to mitochondria upon MCL-1-binding, CHIP mediates ubiquitylation of cytosolic NOXA and promotes lysosomal degradation of NOXA, which is not bound by MCL-1. Our data indicate that MCL-1 defines NOXA abundance and its pro-apoptotic activity. Increased NOXA levels beyond this threshold are effectively removed by lysosomal protein degradation triggered via CHIP-mediated ubiquitylation. Together, these results shed new light on regulatory circuits controlling DNA damage response and identified the E3 ligase CHIP as a new molecular guardian, which restricts the cytosolic accumulation of NOXA upon genotoxic stress.

DOI: https://doi.org/10.1038/s41419-020-02923-x
Biomed Res Int. 2020 ;2020 4834965

The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks.

Lei Zhao, Chengyu Bao, Yuxuan Shang, Xinye He, Chiyuan Ma, Xiaohua Lei, Dong Mi, Yeqing Sun.

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

DOI: https://doi.org/10.1155/2020/4834965
Am J Cancer Res. 2020 ;10(8): 2649-2676

AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers.

Hassan Lemjabbar-Alaoui, Csaba J Peto, Yi-Wei Yang, David M Jablons.

Poly (ADP-ribose) polymerase (PARP) has recently emerged as a central mediator in cancer resistance against numerous anticancer agents to include chemotherapeutic agents such as microtubule targeting agents and DNA damaging agents. Here, we describe AMXI-5001, a novel, highly potent dual PARP1/2 and microtubule polymerization inhibitor with favorable metabolic stability, oral bioavailability, and pharmacokinetic properties. The potency and selectivity of AMXI-5001 were determined by biochemical assays. Anticancer activity either as a single-agent or in combination with other antitumor agents was evaluated in vitro. In vivo antitumor activity as a single-agent was assessed in a triple-negative breast cancer (TNBC) model. AMXI-5001 demonstrates comparable IC50 inhibition against PARP and microtubule polymerization as clinical PARP inhibitors (Olaparib, Rucaparib, Niraparib, and Talazoparib) and the potent polymerization inhibitor (Vinblastine), respectively. In vitro, AMXI-5001 exhibited selective antitumor cytotoxicity across a wide variety of human cancer cells with much lower IC50s than existing clinical PARP1/2 inhibitors. AMXI-5001 is highly active in both BRCA mutated and wild type cancers. AMXI-5001 is orally bioavailable. AMXI-5001 elicited a remarkable In vivo preclinical anti-tumor activity in a BRCA mutated TNBC model. Oral administration of AMXI-5001 induced complete regression of established tumors, including exceedingly large tumors. AMXI-5001 resulted in superior anti-tumor effects compared to either single agent (PARP or microtubule) inhibitor or combination with both agents. AMXI-5001 will enter clinical trial testing soon and represents a promising, novel first in class dual PARP1/2 and microtubule polymerization inhibitor that delivers continuous and synchronous one-two punch cancer therapy with one molecule.

Keywords: AMXI-5001; BRCA; PARP inhibitor; breast cancer; cancer therapy; homologous recombination; malignancy; microtubule inhibitor; synthetic lethality
PLoS One. 2020 ;15(9): e0237981

Cancer proteome and metabolite changes linked to SHMT2.

Jiefei Tong, Jonathan R Krieger, Paul Taylor, Rick Bagshaw, Jae Kang, Swathi Jeedigunta, Leanne E Wybenga-Groot, Wen Zhang, Heba Badr, Shideh Mirhadi, Nhu-An Pham, Étienne Coyaud, Man Yu, Ming Li, Michael Cabanero, Brian Raught, Jason T Maynes, Cynthia Hawkins, Ming Sound Tsao, Michael F Moran.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.

DOI: https://doi.org/10.1371/journal.pone.0237981
J Biol Chem. 2020 Sep 10. pii: jbc.RA120.015699. [Epub ahead of print]

Pif1, RPA and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis.

Melanie A Sparks, Peter M Burgers, Roberto Galletto.

  Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. While the block imparted by Tbf1 can be overcome by the DNA binding activity of the ssDNA binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during Break Induced Replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.

Keywords:  DNA helicase; DNA polymerase; DNA replication; nucleosome; transcription factor

DOI:  https://doi.org/10.1074/jbc.RA120.015699
Int J Mol Sci. 2020 Sep 09. pii: E6602. [Epub ahead of print]21(18):

Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells.

Stefan J Roobol, Irene van den Bent, Wiggert A van Cappellen, Tsion E Abraham, Maarten W Paul, Roland Kanaar, Adriaan B Houtsmuller, Dik C van Gent, Jeroen Essers.

  High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.

Keywords:  DNA double-strand breaks; alpha particles; high linear energy transfer; homologous recombination; live-cell microscopy; nonhomologous DNA end-joining

DOI:  https://doi.org/10.3390/ijms21186602
Proc Natl Acad Sci U S A. 2020 Sep 09. pii: 202007437. [Epub ahead of print]

Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch.

Christl Gaubitz, Xingchen Liu, Joseph Magrino, Nicholas P Stone, Jacob Landeck, Mark Hedglin, Brian A Kelch.

  DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

Keywords:  AAA+; ATPase; DNA replication; clamp loader; sliding clamp

DOI:  https://doi.org/10.1073/pnas.2007437117
Mol Cell. 2020 Sep 01. pii: S1097-2765(20)30577-3. [Epub ahead of print]

Processive Activity of Replicative DNA Polymerases in the Replisome of Live Eukaryotic Cells.

Nitin Kapadia, Ziad W El-Hajj, Huan Zheng, Thomas R Beattie, Angela Yu, Rodrigo Reyes-Lamothe.

  DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.

Keywords:  DNA polymerase; DNA replication; Saccharomyces cerevisiae; budding yeast; live-cell imaging; replisome; single-molecule microscopy

DOI:  https://doi.org/10.1016/j.molcel.2020.08.014
Clin Transl Sci. 2020 Sep 09.

DNA Methylation-Based Epigenetic Repression of SLC22A4 Promotes Resistance to Cytarabine in Acute Myeloid Leukemia.

Daelynn R Buelow, Jason T Anderson, Stanley B Pounds, Lei Shi, Jatinder K Lamba, Shuiying Hu, Alice A Gibson, Emily A Goodwin, Alex Sparreboom, Sharyn D Baker.

Reduced expression of the uptake transporter, OCTN1 (SLC22A4), has been reported as a strong predictor of poor event-free and overall survival in multiple cohorts of patients with acute myeloid leukemia (AML) receiving the cytidine nucleoside analog, cytarabine (Ara-C). To further understand the mechanistic basis of interindividual variability in the functional expression of OCTN1 in AML, we hypothesized a mechanistic connection to DNA methylation-based epigenetic repression of SLC22A4. We found increased basal SLC22A4 methylation was associated with decreased Ara-C uptake in AML cell lines. Pre-treatment with hypomethylating agents, 5-azacytidine, or decitabine, restored SLC22A4 mRNA expression, increased cellular uptake of Ara-C, and was associated with increased cellular sensitivity to Ara-C compared with vehicle-treated cells. Additionally, lower SLC22A4 methylation status was associated with distinct clinical advantages in both adult and pediatric patients with AML. These findings suggest a regulatory mechanism is involved in the interindividual variability in response to Ara-C, and provides a basis for the integration of hypomethylating agents into Ara-C-based treatment regimens.

DOI: https://doi.org/10.1111/cts.12861
Nucleic Acids Res. 2020 Sep 07. pii: gkaa733. [Epub ahead of print]

Hedgehog signaling enables repair of ribosomal DNA double-strand breaks.

Tshering D Lama-Sherpa, Victor T G Lin, Brandon J Metge, Shannon E Weeks, Dongquan Chen, Rajeev S Samant, Lalita A Shevde.

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

DOI: https://doi.org/10.1093/nar/gkaa733
Front Bioeng Biotechnol. 2020 ;8 854

Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.

Maryke Fehlau, Felix Kaspar, Katja F Hellendahl, Julia Schollmeyer, Peter Neubauer, Anke Wagner.

  Nucleoside-5'-triphosphates (NTPs) and their analogs are building blocks of DNA and are important compounds in both pharmaceutical and molecular biology applications. Currently, commercially available base or sugar modified NTPs are mainly synthesized chemically. Since the chemical production of NTPs is time-consuming and generally inefficient, alternative approaches are under development. Here we present a simple, efficient and generalizable enzymatic synthesis method for the conversion of nucleosides to NTPs. Our one-pot method is modular, applicable to a wide range of natural and modified nucleotide products and accesses NTPs directly from cheap nucleoside precursors. Nucleoside kinases, nucleoside monophosphate (NMP) kinases and a nucleoside diphosphate (NDP) kinase were applied as biocatalysts. Enzymes with different substrate specificities were combined to produce derivatives of adenosine and cytidine triphosphate with conversions of 4 to 26%. The implementation of a (deoxy)ATP recycling system resulted in a significant increase in the conversion to all NTP products, furnishing 4 different NTPs in quantitative conversion. Natural (deoxy)NTPs were synthesized with 60 to >99% conversion and sugar- and base-modified NTPs were produced with 69 to >99% and 27 to 75% conversion, respectively. The presented method is suitable for the efficient synthesis of a wide range of natural and modified NTPs in a sustainable one-pot process.

Keywords:  ATP regeneration system; enzymatic cascade synthesis; modular; nucleoside kinase; nucleoside-5′-triphosphate; nucleotide analog; nucleotide kinase; one-pot multi-enzyme reaction

DOI:  https://doi.org/10.3389/fbioe.2020.00854
J Exp Bot. 2020 Sep 09. pii: eraa410. [Epub ahead of print]

TOR mediates the autophagy response to altered nucleotide homeostasis in a ribonuclease mutant.

Zakayo Kazibwe, Junmarie Soto-Burgos, Gustavo C MacIntosh, Diane C Bassham.

  The Arabidopsis thaliana T2 family endoribonuclease RNS2 localizes to the vacuole and functions in rRNA degradation. Loss of RNS2 activity impairs rRNA turnover and leads to constitutive autophagy, a process for degradation of cellular components. Autophagy is normally activated during environmental stress and is important for stress tolerance and homeostasis. Here we show that restoration of cytosolic purine nucleotide levels rescues the constitutive autophagy phenotype of rns2-2 seedlings, whereas inhibition of purine synthesis induces autophagy in wild-type seedlings. rns2-2 seedlings have reduced activity of the target of rapamycin (TOR) kinase complex, a negative regulator of autophagy, and this phenotype is rescued by addition of inosine to increase purine levels. Activation of TOR in rns2-2 by exogenous auxin blocks the enhanced autophagy, indicating a possible involvement of the TOR signaling pathway in the activation of autophagy in the rns2-2 mutant. Our data suggest a model in which loss of rRNA degradation in rns2-2 leads to a reduction in cytoplasmic nucleotide concentrations, which in turn inhibits TOR activity, leading to activation of autophagy to restore homeostasis.

Keywords:  Arabidopsis; RNS2; TOR; autophagy; inosine; nucleotides; rRNA; ribonuclease

DOI:  https://doi.org/10.1093/jxb/eraa410
Sci Adv. 2020 Sep;pii: eabb2630. [Epub ahead of print]6(37):

Extracellular matrix stiffness determines DNA repair efficiency and cellular sensitivity to genotoxic agents.

Min Deng, Jing Lin, Somaira Nowsheen, Tongzheng Liu, Yingchun Zhao, Peter W Villalta, Delphine Sicard, Daniel J Tschumperlin, SeungBaek Lee, JungJin Kim, Zhenkun Lou.

DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. These lesions also contribute to the efficacy of radiotherapy and many cancer chemotherapeutics. DNA repair efficiency is regulated by both intracellular and extracellular chemical signals. However, it is largely unknown whether this process is regulated by physical stimuli such as extracellular mechanical signals. Here, we report that DSB repair is regulated by extracellular mechanical signals. Low extracellular matrix (ECM) stiffness impairs DSB repair and renders cells sensitive to genotoxic agents. Mechanistically, we found that the MAP4K4/6/7 kinases are activated and phosphorylate ubiquitin in cells at low stiffness. Phosphorylated ubiquitin impairs RNF8-mediated ubiquitin signaling at DSB sites, leading to DSB repair deficiency. Our results thus demonstrate that ECM stiffness regulates DSB repair efficiency and genotoxic sensitivity through MAP4K4/6/7 kinase-mediated ubiquitin phosphorylation, providing a previously unidentified regulation in DSB-induced ubiquitin signaling.

DOI: https://doi.org/10.1126/sciadv.abb2630
Am J Physiol Endocrinol Metab. 2020 Sep 07.

New insights into purine metabolism in metabolic diseases: role of xanthine oxidoreductase activity.

Masato Furuhashi.

  Xanthine oxidoreductase (XOR) consists of two different forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), and is a rate-limiting enzyme of uric acid production from hypoxanthine and xanthine. Uric acid is the end-product of purine metabolism in humans and has a powerful antioxidant effect. The lack of ascorbic acid, known as vitamin C, in hominoids has been thought to cause a compensatory increase in uric acid as an antioxidant by unfunctional gene mutation of uricase to a pseudogene. Since XO is involved in an increase in reactive oxygen species (ROS) by generating superoxide and hydrogen peroxide, inadequate activation of XOR promotes oxidative stress-related tissue injury. Plasma XOR activity is associated with obesity, smoking, liver dysfunction, hyperuricemia, dyslipidemia, insulin resistance and adipokines, indicating a novel biomarker of metabolic disorders. However, XOR activity in adipose tissue is low in humans unlike in rodents, and hypoxanthine is secreted from human adipose tissue. The concentration of hypoxanthine, but not xanthine, is independently associated with obesity in a general population, indicating differential regulation of hypoxanthine and xanthine. Treatment with an XOR inhibitor can decrease uric acid for preventing gout, reduce production of XO-related ROS and promote reutilization of hypoxanthine and ATP production through the salvage pathway. It has recently been suggested that discontinuation of an XOR inhibitor causes adverse cardiovascular outcomes as XOR inhibitor withdrawal syndrome, possibly due to cardiac disturbance of conduction and contraction by reduced ATP production. New insights into purine metabolism including the role of XOR activity are discussed in this review.

Keywords:  purine metabolism; uric acid; xanthine dehydrogenase; xanthine oxidase

DOI:  https://doi.org/10.1152/ajpendo.00378.2020
Expert Rev Hematol. 2020 Sep 07.

Treatment of hairy cell leukemia.

Dai Chihara, Robert J Kreitman.

   INTRODUCTION: Purine analogs made dramatic improvements for patients with hairy cell leukemia (HCL), but patients often relapse, require multiple treatments, and may become refractory. Major developments in treatment of relapsed/refractory HCL occurred with discovery of disease biology. New agents increase the complexity of clinical decision-making.
AREAS COVERED: Anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox (Moxe), CD20 Mabs rituximab and obinutuzumab, BRAF/MEK inhibitors vemurafenib and dabrafenib-trametinib, and Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have been tested for HCL. All show efficacy but with different treatment durations and response rates, including for eradicating minimal residual disease (MRD). Side effects differ and must be considered when selecting treatment. Studies from PubMed indexed papers and abstracts presented at major international conferences are included.
EXPERT OPINION: Rituximab with purine analog or BRAF-inhibitor achieves high rates of MRD-free complete remission (CR). Moxe achieves MRD-free CR without chemotherapy toxicities. Moxe should be considered prior to splenectomy or development of adenopathy. BRAF/MEK inhibition and ibrutinib are effective options but most patients remain MRD+, requiring indefinite treatment or rituximab to prevent relapse. Under investigation is MRD elimination with CD20 antibody combined with Moxe or BRAF inhibitor. High-risk disease including HCL variant and IGHV4-34+ unmutated HCL require further investigation. (197 words).

Keywords:  BRAF; CD20; CD22; Hairy cell leukemia; Obinutuzumab; Purine analog; ibrutinib; moxetumomab pasudotox; recombinant immunotoxin; rituximab

DOI:  https://doi.org/10.1080/17474086.2020.1819231