bims-numges 2021-07-11 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–07–11
28 papers selected by
Sean Rudd, Karolinska Institutet

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.
Suppression of DNA Polymerase β Activity Is Synthetically Lethal in BRCA1-Deficient Cells.
Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication.
DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination.
Non-homologous end-joining at challenged replication forks: an RNA connection?
VLX600 Disrupts Homologous Recombination and Synergizes with PARP Inhibitors and Cisplatin by Inhibiting Histone Lysine Demethylases.
The stabilized Pol31-Pol3 interface counteracts Pol32 ablation with differential effects on repair.
Toward Greater Precision in Cancer Radiotherapy.
Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover.
MT1-MMP-dependent ECM processing regulates laminB1 stability and mediates replication fork restart.
NuA4 and SAGA acetyltransferase complexes cooperate for repair of DNA breaks by homologous recombination.
Effects of Chemopreventive Natural Compounds on the Accuracy of 8-oxo-7,8-dihydro-2'-deoxyguanosine Translesion Synthesis.
Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide.
Iron Chelator Transmetalative Approach to Inhibit Human Ribonucleotide Reductase.
DNA Repair Protein Expression and Oxidative/Nitrosative Stress in Ulcerative Colitis and Sporadic Colorectal Cancer.
Role of phase partitioning in coordinating DNA damage response: focus on the Apurinic Apyrimidinic Endonuclease 1 interactome.
A structural framework for DNA replication and transcription through chromatin.
Nuclear Transglutaminase 2 interacts with topoisomerase II⍺ to promote DNA damage repair in lung cancer cells.
DNA-RNA hybrids at DSBs interfere with repair by homologous recombination.
DNA repair glycosylase hNEIL1 triages damaged bases via competing interaction modes.
Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer.
A MYBL2 complex for RRM2 transactivation and the synthetic effect of MYBL2 knockdown with WEE1 inhibition against colorectal cancer.
DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy.
NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir.
PIAS1 potentiates the anti-EBV activity of SAMHD1 through SUMOylation.
FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.
Exosomal IDH1 increases the resistance of colorectal cancer cells to 5-Fluorouracil.
Thioredoxin reductase is a major regulator of metabolism in leukemia cells.

Mol Cell. 2021 Jun 26. pii: S1097-2765(21)00458-5. [Epub ahead of print]

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.

Ke Cong, Min Peng, Arne Nedergaard Kousholt, Wei Ting C Lee, Silviana Lee, Sumeet Nayak, John Krais, Pamela S VanderVere-Carozza, Katherine S Pawelczak, Jennifer Calvo, Nicholas J Panzarino, Jos Jonkers, Neil Johnson, John J Turchi, Eli Rothenberg, Sharon B Cantor.

  Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

Keywords:  BRCA1/BRCA2; Fanconi anemia (FA); Okazaki fragment processing; PARP inhibitor; fork protection; homologous recombination; parylation; replication gaps; ssDNA; synthetic lethal

DOI:  https://doi.org/10.1016/j.molcel.2021.06.011
ACS Chem Biol. 2021 Jul 09.

Suppression of DNA Polymerase β Activity Is Synthetically Lethal in BRCA1-Deficient Cells.

Shelby C Yuhas, Alok Mishra, Theodore L DeWeese, Marc M Greenberg.

People whose cells express mutated forms of the BRCA1 tumor suppressor are at a higher risk for developing cancer. BRCA1-deficient cells are defective in DNA double-strand break repair. The inhibition of poly(ADP-ribose) polymerase 1 in such cells is a synthetically lethal, cytotoxic effect that has been exploited to produce anticancer drugs such as Olaparib. However, alternative synthetic lethal approaches are necessary. We report that DNA polymerase β (Pol β) forms a synthetically lethal interaction with BRCA1. The SiRNA knockdown of Pol β or the treatment with a Pol β pro-inhibitor (pro-1) is cytotoxic in BRCA1-deficient ovarian cancer cells. BRCA1-complemented cells are significantly less susceptible to either treatment. pro-1 is also toxic to BRCA1-deficient breast cancer cells, and its toxicity in BRCA1-deficient cells is comparable to that of Olaparib. These experiments establish Pol β as a synthetically lethal target within BRCA1-deficient cells and a potentially useful one for treating cancer.

DOI: https://doi.org/10.1021/acschembio.1c00385
PLoS Genet. 2021 Jul 06. 17(7): e1009526

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication.

Ignacio Soriano, Enrique Vazquez, Nagore De Leon, Sibyl Bertrand, Ellen Heitzer, Sophia Toumazou, Zhihan Bo, Claire Palles, Chen-Chun Pai, Timothy C Humphrey, Ian Tomlinson, Sue Cotterill, Stephen E Kearsey.

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.

DOI: https://doi.org/10.1371/journal.pgen.1009526
Nat Commun. 2021 07 05. 12(1): 4126

DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination.

Prasun Chakraborty, Kevin Hiom.

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

DOI: https://doi.org/10.1038/s41467-021-24341-z
Trends Genet. 2021 Jul 05. pii: S0168-9525(21)00166-9. [Epub ahead of print]

Non-homologous end-joining at challenged replication forks: an RNA connection?

Charlotte Audoynaud, Stéphan Vagner, Sarah Lambert.

  Defective DNA replication, known as 'replication stress', is a source of DNA damage, a hallmark of numerous human diseases, including cancer, developmental defect, neurological disorders, and premature aging. Recent work indicates that non-homologous end-joining (NHEJ) is unexpectedly active during DNA replication to repair replication-born DNA lesions and to safeguard replication fork integrity. However, erroneous NHEJ events are deleterious to genome stability. RNAs are novel regulators of NHEJ activity through their ability to modulate the assembly of repair complexes in trans. At DNA damage sites, RNAs and DNA-embedded ribonucleotides modulate repair efficiency and fidelity. We discuss here how RNAs and associated proteins, including RNA binding proteins, may regulate NHEJ to sustain genome stability during DNA replication.

Keywords:  DNA replication fork; RNA; genome stability; non-homologous end-joining; single- and double-ended double-strand break

DOI:  https://doi.org/10.1016/j.tig.2021.06.010
Mol Cancer Ther. 2021 Jun 17. pii: molcanther.1099.2020. [Epub ahead of print]

VLX600 Disrupts Homologous Recombination and Synergizes with PARP Inhibitors and Cisplatin by Inhibiting Histone Lysine Demethylases.

Thomas L Ekstrom, Nicholas M Pathoulas, Amelia M Huehls, Arun Kanakkanthara, Larry M Karnitz.

Tumors with defective homologous recombination (HR) DNA repair are more sensitive to chemotherapies that induce lesions repaired by HR as well as poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis). However, these therapies have limited activity in HR-proficient cells. Accordingly, agents that disrupt HR may be a means to augment the activities of these therapies in HR-proficient tumors. Here we show that VLX600, a small molecule that has been in a phase I clinical trial, disrupts HR and synergizes with PARPis and platinum compounds in ovarian cancer cells. We further found that VLX600 and other iron chelators disrupt HR, in part, by inhibiting iron-dependent histone lysine demethylases (KDM) family members, thus blocking recruitment of HR repair proteins, including RAD51, to double-strand DNA breaks. Collectively, these findings suggest that pharmacologically targeting KDM family members with VLX600 may be a potential novel strategy to therapeutically induce HR defects in ovarian cancers and correspondingly sensitize them to platinum agents and PARPis, two standard-of-care therapies for ovarian cancer.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-1099
Life Sci Alliance. 2021 Sep;pii: e202101138. [Epub ahead of print]4(9):

The stabilized Pol31-Pol3 interface counteracts Pol32 ablation with differential effects on repair.

Kenji Shimada, Monika Tsai-Pflugfelder, Niloofar Davoodi Vijeh Motlagh, Neda Delgoshaie, Jeannette Fuchs, Heinz Gut, Susan M Gasser.

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3-Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31-Pol3 and Pol31-Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

DOI: https://doi.org/10.26508/lsa.202101138
Cancer Res. 2021 Jun 15. 81(12): 3156-3157

Toward Greater Precision in Cancer Radiotherapy.

Jac A Nickoloff.

The cellular DNA damage response (DDR) is a key factor in tumor suppression and tumor responses to genotoxic chemo- and radiotherapy. Master DDR regulators include three phosphatidyl inositol 3' kinase-related kinases (PIKK) called ATM, ATR, and the catalytic subunit of DNA-dependent protein kinase, DNA-PKcs. Among their many functions, PIKKs regulate repair of DNA double-strand breaks (DSB) by homologous recombination (HR) and nonhomologous end-joining (NHEJ). Ionizing radiation induces DSBs that are either widely dispersed and efficiently repaired, or clustered and poorly repaired by the dominant NHEJ pathway. The inefficient repair of clustered DSBs by NHEJ shifts repair toward the competing HR pathway. In this issue of Cancer Research, Zhou and colleagues revealed a novel synthetic lethal approach in which the greater dependency on HR to repair clustered DSBs induced by protons is exploited to enhance killing of tumor cells and tumor xenografts by suppressing HR with an ATM inhibitor or mutant BRCA1. This is an important step toward precision cancer radiotherapy.See related article by Zhou et al., p. 3333.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0664
Nucleic Acids Res. 2021 Jul 07. pii: gkab591. [Epub ahead of print]

Single molecule analysis indicates stimulation of MUTYH by UV-DDB through enzyme turnover.

Sunbok Jang, Matthew A Schaich, Cindy Khuu, Brittani L Schnable, Chandrima Majumdar, Simon C Watkins, Sheila S David, Bennett Van Houten.

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4-5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.

DOI: https://doi.org/10.1093/nar/gkab591
PLoS One. 2021 ;16(7): e0253062

MT1-MMP-dependent ECM processing regulates laminB1 stability and mediates replication fork restart.

Varsha Thakur, Juliano Tiburcio de Freitas, Yuan Li, Keman Zhang, Alyssa Savadelis, Barbara Bedogni.

Radiotherapy remains a mainstay of treatment for a majority of cancer patients. We have previously shown that the membrane bound matrix metalloproteinase MT1-MMP confers radio- and chemotherapy resistance to breast cancer via processing of the ECM and activation of integrinβ1/FAK signaling. Here, we further discovered that the nuclear envelope protein laminB1 is a potential target of integrinβ1/FAK. FAK interacts with laminB1 contributing to its stability. Stable laminB1 is found at replication forks (RFs) where it is likely to allow the proper positioning of RF protection factors, thus preventing RF degradation. Indeed, restoration of laminB1 expression rescues replication fork stalling and collapse that occurs upon MT1-MMP inhibition, and reduces DNA damage in breast cancer cells. Together, these data highlight a novel mechanism of laminB1 stability and replication fork restart via MT1-MMP dependent extracelluar matrix remodeling.

DOI: https://doi.org/10.1371/journal.pone.0253062
PLoS Genet. 2021 Jul 06. 17(7): e1009459

NuA4 and SAGA acetyltransferase complexes cooperate for repair of DNA breaks by homologous recombination.

Xue Cheng, Valérie Côté, Jacques Côté.

Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination.

DOI: https://doi.org/10.1371/journal.pgen.1009459
Planta Med. 2021 Jul 08.

Effects of Chemopreventive Natural Compounds on the Accuracy of 8-oxo-7,8-dihydro-2'-deoxyguanosine Translesion Synthesis.

Amandine Nachtergael, Déborah Lanterbecq, Martin Spanoghe, Alexandra Belayew, Pierre Duez.

Translesion synthesis is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis. Specialized translesion synthesis DNA polymerases pursue replication by inserting a base opposite to this lesion, correctly or incorrectly depending on the lesion nature, involved DNA polymerase(s), sequence context, and still unknown factors. To measure the correct or mutagenic outcome of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass by translesion synthesis, a primer-extension assay was performed in vitro on a template DNA bearing this lesion in the presence of nuclear proteins extracted from human intestinal epithelial cells (FHs 74 Int cell line); the reaction products were analyzed by both denaturing capillary electrophoresis (to measure the yield of translesion elongation) and pyrosequencing (to determine the identity of the nucleotide inserted in front of the lesion). The influence of 14 natural polyphenols on the correct or mutagenic outcome of translesion synthesis through 8-oxo-7,8-dihydro-2'-deoxyguanosine was then evaluated in 2 experimental conditions by adding the polyphenol either (i) to the reaction mix during the primer extension assay; or (ii) to the culture medium, 24 h before cell harvest and nuclear proteins extraction. Most of the tested polyphenols significantly influenced the outcome of translesion synthesis, either through an error-free (apigenin, baicalein, sakuranetin, and myricetin) or a mutagenic pathway (epicatechin, chalcone, genistein, magnolol, and honokiol).

DOI: https://doi.org/10.1055/a-1527-1435
Elife. 2021 Jul 08. pii: e69544. [Epub ahead of print]10

Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide.

Robert P Fuchs, Asako Isogawa, Joao A Paulo, Kazumitsu Onizuka, Tatsuro Takahashi, Ravindra Amunugama, Julien P Duxin, Shingo Fujii.

  Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-studied cell cycle dependent mode of action.

Keywords:  biochemistry; chemical biology; chromosomes; gene expression; xenopus

DOI:  https://doi.org/10.7554/eLife.69544
JACS Au. 2021 Jun 28. 1(6): 865-878

Iron Chelator Transmetalative Approach to Inhibit Human Ribonucleotide Reductase.

Kavita Gaur, Sofia C Pérez Otero, Josué A Benjamín-Rivera, Israel Rodríguez, Sergio A Loza-Rosas, Alexandra M Vázquez Salgado, Eman A Akam, Liz Hernández-Matias, Rohit K Sharma, Nahiara Alicea, Martin Kowaleff, Anthony V Washington, Andrei V Astashkin, Elisa Tomat, Arthur D Tinoco.

Efforts directed at curtailing the bioavailability of intracellular iron could lead to the development of broad-spectrum anticancer drugs given the metal's role in cancer proliferation and metastasis. Human ribonucleotide reductase (RNR), the key enzyme responsible for synthesizing the building blocks of DNA replication and repair, depends on Fe binding at its R2 subunit to activate the catalytic R1 subunit. This work explores an intracellular iron chelator transmetalative approach to inhibit RNR using the titanium(IV) chemical transferrin mimetic (cTfm) compounds Ti(HBED) and Ti(Deferasirox)2. Whole-cell EPR studies reveal that the compounds can effectively attenuate RNR activity though seemingly causing different changes to the labile iron pool that may account for differences in their potency against cells. Studies of Ti(IV) interactions with the adenosine nucleotide family at pH 7.4 reveal strong metal binding and extensive phosphate hydrolysis, which suggest the capacity of the metal to disturb the nucleotide substrate pool of the RNR enzyme. By decreasing intracellular Fe bioavailability and altering the nucleotide substrate pool, the Ti cTfm compounds could inhibit the activity of the R1 and R2 subunits of RNR. The compounds arrest the cell cycle in the S phase, indicating suppressed DNA replication, and induce apoptotic cell death. Cotreatment cell viability studies with cisplatin and Ti(Deferasirox)2 reveal a promising synergism between the compounds that is likely owed to their distinct but complementary effect on DNA replication.

DOI: https://doi.org/10.1021/jacsau.1c00078
Anticancer Res. 2021 Jul;41(7): 3261-3270

DNA Repair Protein Expression and Oxidative/Nitrosative Stress in Ulcerative Colitis and Sporadic Colorectal Cancer.

Paula M DE Angelis, Linda Dorg, Sean Pham, Solveig Norheim Andersen.

   BACKGROUND/AIM: Chronic inflammation generates large quantities of reactive oxygen and nitrogen species that damage DNA. DNA repair is important for cellular viability and genome integrity.
MATERIALS AND METHODS: Expression levels of the DNA repair proteins OGG1, XPA, MLH1, PARP1, and XRCC6, which function in base excision repair, nucleotide excision repair, mismatch repair, single-strand break repair and double-strand break repair, respectively, were assessed using immunohistochemistry in ulcerative colitis and sporadic colorectal cancer biopsies. Levels of oxidative/ nitrosative stress biomarkers were also assessed.
RESULTS: Ulcerative colitis and colorectal cancer lesions expressed significantly higher levels of all DNA repair proteins and oxidative/ nitrosative stress biomarkers compared to normal colonic mucosa. Ulcerative colitis had the highest XPA and XRCC6 expression.
CONCLUSION: Oxidative/nitrosative stress is prevalent in the colon of both diseases. Nucleotide excision repair and non-homologous end-joining double-strand break repair may be compromised in colorectal cancer, but not in ulcerative colitis.

Keywords:  3-nitrotyrosine; 4-hydroxy-2-nonenal; DNA repair proteins; Ulcerative colitis; immunohistochemistry; oxidative/nitrosative stress biomarkers; sporadic colorectal cancer

DOI:  https://doi.org/10.21873/anticanres.15112
Biomol Concepts. 2020 Dec 23. 11(1): 209-220

Role of phase partitioning in coordinating DNA damage response: focus on the Apurinic Apyrimidinic Endonuclease 1 interactome.

Damiano Tosolini, Giulia Antoniali, Emiliano Dalla, Gianluca Tell.

  Liquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions while excluding noninteracting components. Disordered domains of proteins, as well as interaction with RNA, favor condensation but are not mandatory for modulating this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses, conferring both spatial and temporal fine regulation. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway, bearing additional 'non-canonical' DNA-repair functions associated with processes like RNA metabolism. Recently, it has been highlighted that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in different pathogenic events. This work might represent a paradigmatical pipeline for evaluating the relevance of LLPS in DDR.

Keywords:  Base excision repair; RNA; bioinformatics; interactome; phase-separation

DOI:  https://doi.org/10.1515/bmc-2020-0019
Curr Opin Struct Biol. 2021 Jul 01. pii: S0959-440X(21)00077-4. [Epub ahead of print]71 51-58

A structural framework for DNA replication and transcription through chromatin.

Oliver Willhoft, Alessandro Costa.

In eukaryotic cells, DNA replication and transcription machineries uncoil nucleosomes along the double helix, to achieve the exposure of the single-stranded DNA template for nucleic acid synthesis. The replisome and RNA polymerases then redeposit histones onto DNA behind the advancing molecular motor, in a process that is crucial for epigenetic inheritance and homeostasis, respectively. Here, we compare and contrast the mechanisms by which these molecular machines advance through nucleosome arrays and discuss how chromatin remodellers can facilitate DNA replication and transcription.

DOI: https://doi.org/10.1016/j.sbi.2021.05.008
J Exp Clin Cancer Res. 2021 Jul 05. 40(1): 224

Nuclear Transglutaminase 2 interacts with topoisomerase II⍺ to promote DNA damage repair in lung cancer cells.

Xiao Lei, Kun Cao, Yuanyuan Chen, Hui Shen, Zhe Liu, Hongran Qin, Jianming Cai, Fu Gao, Yanyong Yang.

   BACKGROUND: To block repairs of DNA damages, especially the DNA double strand break (DSB) repair, can be used to induce cancer cell death. DSB repair depends on a sequential activation of DNA repair factors that may be potentially targeted for clinical cancer therapy. Up to now, many protein components of DSB repair complex remain unclear or poorly characterized. In this study, we discovered that Transglutaminase 2 (TG2) acted as a new component of DSB repair complex.
METHODS: A bioinformatic analysis was performed to identify DNA damage relative genes from dataset from The Cancer Genome Atlas. Immunofluorescence and confocal microscopy were used to monitor the protein localization and recruitment kinetics. Furthermore, immunoprecipitation and mass spectrometry analysis were performed to determine protein interaction of both full-length and fragments or mutants in distinct domain. In situ lung cancer model was used to study the effects cancer therapy in vivo.
RESULTS: After DSB induction, cytoplasmic TG2 was extensively mobilized and translocated into nucleus after phosphorylated at T162 site by DNA-PKcs. Nuclear TG2 quickly accumulated at DSB sites and directly interacting with Topoisomerase IIα (TOPOIIα) with its TGase domain to promote DSB repair. TG2 deficient cells lost capacity of DSB repair and become susceptible to ionizing radiation. Specific inhibition of TG2-TOPOIIα interaction by glucosamine also significantly inhibited DSB repair, which increased sensitivity in lung cancer cells and engrafted lung cancers.
CONCLUSIONS: These findings elucidate new mechanism of TG2 in DSB repair trough directly interacting with TOPOIIα, inhibition of which provided potential target for overcoming cancer resistance.

Keywords:  Cancer therapy; DNA damage repair; DNA double strand breaks (DSBs); Topoisomerase IIα (TOPOIIα); Transglutaminase 2 (TG2)

DOI:  https://doi.org/10.1186/s13046-021-02009-2
Elife. 2021 Jul 08. pii: e69881. [Epub ahead of print]10

DNA-RNA hybrids at DSBs interfere with repair by homologous recombination.

Pedro Ortega, Jose Antonio Mérida-Cerro, Ana G Rondón, Belén Gómez-González, Andrés Aguilera.

  DNA double strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with recombinational repair we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription, and need to be removed to allow recombinational repair, rather than playing a positive role.

Keywords:  S. cerevisiae; chromosomes; gene expression

DOI:  https://doi.org/10.7554/eLife.69881
Nat Commun. 2021 07 05. 12(1): 4108

DNA repair glycosylase hNEIL1 triages damaged bases via competing interaction modes.

Menghao Liu, Jun Zhang, Chenxu Zhu, Xiaoxue Zhang, Weide Xiao, Yongchang Yan, Lulu Liu, Hu Zeng, Yi Qin Gao, Chengqi Yi.

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.

DOI: https://doi.org/10.1038/s41467-021-24431-y
Int J Biol Sci. 2021 ;17(9): 2240-2251

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer.

Thuy Phan, Vu H Nguyen, Ralf Buettner, Corey Morales, Lifeng Yang, Paul Wong, Weiman Tsai, Marcela d'Alincourt Salazar, Ziv Gil, Don J Diamond, Joshua D Rabinowitz, Steven Rosen, Laleh G Melstrom.

  Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer. Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling. Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05). Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.

Keywords:  de novo pyrimidine synthesis; leflunomide; pancreatic cancer

DOI:  https://doi.org/10.7150/ijbs.60473
Cell Death Dis. 2021 Jul 07. 12(7): 683

A MYBL2 complex for RRM2 transactivation and the synthetic effect of MYBL2 knockdown with WEE1 inhibition against colorectal cancer.

Qian Liu, Lijuan Guo, Hongyan Qi, Meng Lou, Rui Wang, Boning Hai, Kailun Xu, Lijun Zhu, Yongfeng Ding, Chen Li, Lingdan Xie, Jing Shen, Xueping Xiang, Jimin Shao.

Ribonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

DOI: https://doi.org/10.1038/s41419-021-03969-1
Signal Transduct Target Ther. 2021 Jul 09. 6(1): 254

DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy.

Ruixue Huang, Ping-Kun Zhou.

Genomic instability is the hallmark of various cancers with the increasing accumulation of DNA damage. The application of radiotherapy and chemotherapy in cancer treatment is typically based on this property of cancers. However, the adverse effects including normal tissues injury are also accompanied by the radiotherapy and chemotherapy. Targeted cancer therapy has the potential to suppress cancer cells' DNA damage response through tailoring therapy to cancer patients lacking specific DNA damage response functions. Obviously, understanding the broader role of DNA damage repair in cancers has became a basic and attractive strategy for targeted cancer therapy, in particular, raising novel hypothesis or theory in this field on the basis of previous scientists' findings would be important for future promising druggable emerging targets. In this review, we first illustrate the timeline steps for the understanding the roles of DNA damage repair in the promotion of cancer and cancer therapy developed, then we summarize the mechanisms regarding DNA damage repair associated with targeted cancer therapy, highlighting the specific proteins behind targeting DNA damage repair that initiate functioning abnormally duo to extrinsic harm by environmental DNA damage factors, also, the DNA damage baseline drift leads to the harmful intrinsic targeted cancer therapy. In addition, clinical therapeutic drugs for DNA damage and repair including therapeutic effects, as well as the strategy and scheme of relative clinical trials were intensive discussed. Based on this background, we suggest two hypotheses, namely "environmental gear selection" to describe DNA damage repair pathway evolution, and "DNA damage baseline drift", which may play a magnified role in mediating repair during cancer treatment. This two new hypothesis would shed new light on targeted cancer therapy, provide a much better or more comprehensive holistic view and also promote the development of new research direction and new overcoming strategies for patients.

DOI: https://doi.org/10.1038/s41392-021-00648-7
Nat Commun. 2021 Jul 07. 12(1): 4181

NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir.

Rina Nishii, Takanori Mizuno, Daniel Rehling, Colton Smith, Brandi L Clark, Xujie Zhao, Scott A Brown, Brandon Smart, Takaya Moriyama, Yuji Yamada, Tatsuo Ichinohe, Makoto Onizuka, Yoshiko Atsuta, Lei Yang, Wenjian Yang, Paul G Thomas, Pål Stenmark, Motohiro Kato, Jun J Yang.

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.

DOI: https://doi.org/10.1038/s41467-021-24509-7
Cell Biosci. 2021 Jul 08. 11(1): 127

PIAS1 potentiates the anti-EBV activity of SAMHD1 through SUMOylation.

Farjana Saiada, Kun Zhang, Renfeng Li.

   BACKGROUND: Sterile alpha motif and HD domain 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase (dNTPase) that restricts the infection of a variety of RNA and DNA viruses, including herpesviruses. The anti-viral function of SAMHD1 is associated with its dNTPase activity, which is regulated by several post-translational modifications, including phosphorylation, acetylation and ubiquitination. Our recent studies also demonstrated that the E3 SUMO ligase PIAS1 functions as an Epstein-Barr virus (EBV) restriction factor. However, whether SAMHD1 is regulated by PIAS1 to restrict EBV replication remains unknown.
RESULTS: In this study, we showed that PIAS1 interacts with SAMHD1 and promotes its SUMOylation. We identified three lysine residues (K469, K595 and K622) located on the surface of SAMHD1 as the major SUMOylation sites. We demonstrated that phosphorylated SAMHD1 can be SUMOylated by PIAS1 and SUMOylated SAMHD1 can also be phosphorylated by viral protein kinases. We showed that SUMOylation-deficient SAMHD1 loses its anti-EBV activity. Furthermore, we demonstrated that SAMHD1 is associated with EBV genome in a PIAS1-dependent manner.
CONCLUSION: Our study reveals that PIAS1 synergizes with SAMHD1 to inhibit EBV lytic replication through protein-protein interaction and SUMOylation.

Keywords:  Cytomegalovirus; Deoxynucleotide triphosphohydrolase; Epstein-Barr virus; Herpesvirus; PIAS1; Phosphorylation; Restriction factor; SAMHD1; SUMOylation

DOI:  https://doi.org/10.1186/s13578-021-00636-y
Mol Cell Biol. 2021 Jul 06. MCB0030321

FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.

Katja Kratz, Mariela Artola-Borán, Saho Kobayashi-Era, Gene Koh, Goncalo Oliveira, Shunsuke Kobayashi, Andreia Oliveira, Xueqing Zou, Julia Richter, Masataka Tsuda, Hiroyuki Sasanuma, Shunichi Takeda, Joanna I Loizou, Alessandro A Sartori, Serena Nik-Zainal, Josef Jiricny.

Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1 and PMS2 are linked to cancer of the colon and other organs, characterised by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1, encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes towards MMR in vivo: cells lacking both EXO1 and FAN1 have a MMR defect and display resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, implying that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

DOI: https://doi.org/10.1128/MCB.00303-21
J Cancer. 2021 ;12(16): 4862-4872

Exosomal IDH1 increases the resistance of colorectal cancer cells to 5-Fluorouracil.

Hao Yang, Sha Xie, Beibei Liang, Qiqi Tang, Huanchen Liu, Dongliang Wang, Gang Huang.

  Chemoresistance challenges the clinical treatment of colorectal cancer and requires an urgent solution. Isocitrate dehydrogenase 1 (IDH1) is a key enzyme involved in glucose metabolism that mediates the malignant transformation of tumors. However, the mechanisms by which IDH1 is involved in colorectal cancer cell proliferation and drug resistance induction remain unclear. In this study, we found that IDH1 was highly expressed in human colorectal cancer tissues and could be used to indicate a high-grade tumor. In vitro gene overexpression and knockdown were used to determine whether IDH1 promoted the proliferation of the colorectal cancer cell line HCT8 and resistance to 5-Fluorouracil (5FU). Further studies have shown that the 5FU-resistant cell line, HCT8FU, secreted exosomes that contained a high level of IDH1 protein. The exosomal IDH1 derived from 5FU-resistant cells enhanced the resistance of 5FU-sensitive cells. Metabolic assays revealed that exosomes derived from 5FU-resistant cells promoted a decrease in the level of IDH1-mediated NADPH, which is associated with the development of 5FU resistance in colorectal cancer cells. Therefore, exosomal IDH1 may be the transmitter and driver of chemoresistance in colorectal cancer and a potential chemotherapy target.

Keywords:  5-Fluorouracil; IDH1; colorectal cancer; exosomes

DOI:  https://doi.org/10.7150/jca.58846
Oncogene. 2021 Jul 08.

Thioredoxin reductase is a major regulator of metabolism in leukemia cells.

Sheelarani Karunanithi, Ruifu Liu, Yongchun Hou, Giancarlo Gonzalez, Natasha Oldford, Anne Jessica Roe, Nethrie Idipilly, Kalpana Gupta, Chandra Sekhar Amara, Satwikreddy Putluri, Grace Kyueun Lee, Juan Valentin-Goyco, Lindsay Stetson, Stephen A Moreton, Vasanta Putluri, Shyam M Kavuri, Yogen Saunthararajah, Marcos de Lima, Gregory P Tochtrop, Nagireddy Putluri, David N Wald.

Despite the fact that AML is the most common acute leukemia in adults, patient outcomes are poor necessitating the development of novel therapies. We identified that inhibition of Thioredoxin Reductase (TrxR) is a promising strategy for AML and report a highly potent and specific inhibitor of TrxR, S-250. Both pharmacologic and genetic inhibition of TrxR impairs the growth of human AML in mouse models. We found that TrxR inhibition leads to a rapid and marked impairment of metabolism in leukemic cells subsequently leading to cell death. TrxR was found to be a major and direct regulator of metabolism in AML cells through impacts on both glycolysis and the TCA cycle. Studies revealed that TrxR directly regulates GAPDH leading to a disruption of glycolysis and an increase in flux through the pentose phosphate pathway (PPP). The combined inhibition of TrxR and the PPP led to enhanced leukemia growth inhibition. Overall, TrxR abrogation, particularly with S-250, was identified as a promising strategy to disrupt AML metabolism.

DOI: https://doi.org/10.1038/s41388-021-01924-0