bims-numges 2022-06-19 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–06–19
27 papers selected by
Sean Rudd, Karolinska Institutet

Hallmarks of DNA replication stress.
Obstacles and opportunities for base excision repair in chromatin.
Dynamic interaction of BRCA2 with telomeric G-quadruplexes underlies telomere replication homeostasis.
PD-L1 deficiency sensitizes tumor cells to DNA-PK inhibition and enhances cGAS-STING activation.
Structural features of DNA polymerases β and λ in complex with benzo[a]pyrene-adducted DNA cause a difference in lesion tolerance.
Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy.
Mechanism of replication origin melting nucleated by CMG helicase assembly.
Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR-based screens.
Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response.
Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae.
Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM.
Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer.
The Pathogenic R3052W BRCA2 Variant Disrupts Homology-Directed Repair by Failing to Localize to the Nucleus.
Efficient discrimination against RNA-containing primers by human DNA polymerase ε.
Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania.
Targeting the Leukemic stem cell protein machinery by inhibition of mitochondrial pyrimidine synthesis.
Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage.
XPC multifaceted roles beyond DNA damage repair: p53-dependent and p53-independent functions of XPC in cell fate decisions.
Mutagenic repair during antibody diversification: emerging insights.
Chk1 inhibitor-induced DNA damage increases BFL1 and decreases BIM but does not protect human cancer cell lines from Chk1 inhibitor-induced apoptosis.
Targeting the DNA repair enzymes MTH1 and OGG1 as a novel approach to treat inflammatory diseases.
The first structure of human MTHFD2L and its implications for the development of isoform-selective inhibitors.
ZFP281-BRCA2 prevents R-loop accumulation during DNA replication.
Remarkable Response of Hairy Cell Leukemia Variant to Single-Agent Cladribine.
What's next for the synthetic lethality drug discovery engine?
Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo.
A pan-cancer compendium of chromosomal instability.

Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00438-5. [Epub ahead of print]82(12): 2298-2314

Hallmarks of DNA replication stress.

Sneha Saxena, Lee Zou.

  Faithful DNA replication is critical for the maintenance of genomic integrity. Although DNA replication machinery is highly accurate, the process of DNA replication is constantly challenged by DNA damage and other intrinsic and extrinsic stresses throughout the genome. A variety of cellular stresses interfering with DNA replication, which are collectively termed replication stress, pose a threat to genomic stability in both normal and cancer cells. To cope with replication stress and maintain genomic stability, cells have evolved a complex network of cellular responses to alleviate and tolerate replication problems. This review will focus on the major sources of replication stress, the impacts of replication stress in cells, and the assays to detect replication stress, offering an overview of the hallmarks of DNA replication stress.

Keywords:  ATR; DNA damage; DNA repair; DNA replication; cancer; cell cycle; checkpoint; genomic integrity; genomic stability; replication stress

DOI:  https://doi.org/10.1016/j.molcel.2022.05.004
DNA Repair (Amst). 2022 May 28. pii: S1568-7864(22)00078-7. [Epub ahead of print]116 103345

Obstacles and opportunities for base excision repair in chromatin.

Dana J Biechele-Speziale, Treshaun B Sutton, Sarah Delaney.

  Most eukaryotic DNA is packaged into chromatin, which is made up of tandemly repeating nucleosomes. This packaging of DNA poses a significant barrier to the various enzymes that must act on DNA, including DNA damage response enzymes that interact intimately with DNA to prevent mutations and cell death. To regulate access to certain DNA regions, chromatin remodeling, variant histone exchange, and histone post-translational modifications have been shown to assist several DNA repair pathways including nucleotide excision repair, single strand break repair, and double strand break repair. While these chromatin-level responses have been directly linked to various DNA repair pathways, how they modulate the base excision repair (BER) pathway remains elusive. This review highlights recent findings that demonstrate how BER is regulated by the packaging of DNA into nucleosome core particles (NCPs) and higher orders of chromatin structures. We also summarize the available data that indicate BER may be enabled by chromatin modifications and remodeling.

Keywords:  Base excision repair; Chromatin; Nucleosome core particle

DOI:  https://doi.org/10.1016/j.dnarep.2022.103345
Nat Commun. 2022 Jun 13. 13(1): 3396

Dynamic interaction of BRCA2 with telomeric G-quadruplexes underlies telomere replication homeostasis.

Junyeop Lee, Keewon Sung, So Young Joo, Jun-Hyeon Jeong, Seong Keun Kim, Hyunsook Lee.

BRCA2-deficient cells precipitate telomere shortening upon collapse of stalled replication forks. Here, we report that the dynamic interaction between BRCA2 and telomeric G-quadruplex (G4), the non-canonical four-stranded secondary structure, underlies telomere replication homeostasis. We find that the OB-folds of BRCA2 binds to telomeric G4, which can be an obstacle during replication. We further demonstrate that BRCA2 associates with G-triplex (G3)-derived intermediates, which are likely to form during direct interconversion between parallel and non-parallel G4. Intriguingly, BRCA2 binding to G3 intermediates promoted RAD51 recruitment to the telomere G4. Furthermore, MRE11 resected G4-telomere, which was inhibited by BRCA2. Pathogenic mutations at the OB-folds abrogated the binding with telomere G4, indicating that the way BRCA2 associates with telomere is innate to its tumor suppressor activity. Collectively, we propose that BRCA2 binding to telomeric G4 remodels it and allows RAD51-mediated restart of the G4-driven replication fork stalling, simultaneously preventing MRE11-mediated breakdown of telomere.

DOI: https://doi.org/10.1038/s41467-022-31156-z
Am J Cancer Res. 2022 ;12(5): 2363-2375

PD-L1 deficiency sensitizes tumor cells to DNA-PK inhibition and enhances cGAS-STING activation.

Zhen Xue, Shuang Zheng, Dongli Linghu, Boning Liu, Yi Yang, Mei-Kuang Chen, Hua Huang, Jiaming Song, Hongyue Li, Jing Wang, Mien-Chie Hung, Diansheng Zhong, Linlin Sun.

Immunotherapies that block PD-L1/PD-1 immune checkpoint proteins represent a landmark breakthrough in cancer treatment. Although the role of PD-L1 in suppressing T cell activity has been extensively studied, its cancer cell-intrinsic functions are not well understood. Herein, we demonstrated that PD-L1 is important for the repair of DNA damage in cancer cells. Mechanically, depletion of PD-L1 led to the downregulation of the critical molecules involved in the homologous recombination (HR) repair pathway, such as ATM and BRCA1, but did not obviously affect the non-homologous end joining (NHEJ) pathway. Notably, PD-L1 silence sensitized cancer cells to chemotherapy agents and the inhibitor of DNA-PK, which is an important kinase for NHEJ. Furthermore, PD-L1 depletion potentiated DNA damage-induced cGAS-STING pathway and induction of IFNβ. The regulation of DNA repair and cGAS-STING pathway by PD-L1 represents its connection with innate immunity that can be exploited to enhance the efficacy of existing immunotherapy. Our findings thus expand the focus of PD-L1 from tumor antigen-specific CD8+ T cells to innate immunity, and support targeting tumor-intrinsic PD-L1 combined with DNA-PK inhibition for tumor eradication, through promoting synthetic lethality and innate immune response.

Keywords: DNA damage response; Immune checkpoint; homologous recombination; innate immunity; non-homologous end joining; synthetic lethality
DNA Repair (Amst). 2022 Jun 03. pii: S1568-7864(22)00086-6. [Epub ahead of print]116 103353

Structural features of DNA polymerases β and λ in complex with benzo[a]pyrene-adducted DNA cause a difference in lesion tolerance.

Nadejda I Rechkunova, Polina V Zhdanova, Natalia A Lebedeva, Ekaterina A Maltseva, Vladimir V Koval, Olga I Lavrik.

  DNA polymerases β (Pol β) and λ (Pol λ) belong to one structural family (X family) and possess the same enzymatic activities. Nonetheless, these enzymes have differences in their catalytic efficiency and specificity. We have previously reported that these enzymes can bypass bulky benzo[a]pyrene-DNA adducts via translesion synthesis during gap-filling reactions, although efficiency and specificity are dependent on the reaction conditions and adduct conformation. In the present study, we analyzed structural features of Pols β and λ complexed with a gapped DNA duplex containing either cis- or trans-benzo[a]pyrene-diol epoxide-N2-dG (BP-dG) using molecular dynamics simulations. It was found that the most pronounced structural difference lies in the positioning of the trans-BP-dG residue relative to secondary structures of the protein; this dissimilarity may explain the differences between Pols β and λ in gap-filling/translesion synthesis. In the case of Pol β, trans-BP-dG turned out to be positioned parallel to the α-helix and β-sheet. In the Pol λ complex, trans-BP-dG is perpendicular to the α-helix. This difference persisted throughout the molecular dynamics trajectory. Selectivity for the BP-dG isomers remained after a deletion of noncatalytic domains of Pol λ. Modeling of Pol λ or β complexes with cis-BP-dG-containing DNA in the presence of Mn2+ either at both metal-binding sites or at the catalytic site only revealed that for both enzymes, the model of the complex containing both Mg2+ and Mn2+ is stabler than that containing two Mn2+ ions. This observation may reflect a shared property of these enzymes: the preference for Mn2+ in terms of catalysis and for Mg2+ regarding triphosphate coordination during the translesion reaction.

Keywords:  Base excision repair; Benzo[a]pyrene; DNA polymerase; Lesion bypass; Molecular dynamics

DOI:  https://doi.org/10.1016/j.dnarep.2022.103353
Methods Mol Biol. 2022 ;2528 1-20

Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy.

Henriette Stoy, Joel Luethi, Fabienne K Roessler, Johannes Riemann, Andres Kaech, Massimo Lopes.

  R-loop are physiologically present on genomic DNA of different organisms and play important roles in genome regulation. However, an increase in their abundance and/or size has been suggested to interfere with the DNA replication process, contributing to genome instability. Most available approaches to monitor R-loops are based on antibodies/enzymes that cannot effectively distinguish R-loops from DNA-RNA hybrids and assess R-loop size and frequency in a population of molecules. Electron microscopy has successfully allowed single-molecule visualization of DNA replication and repair intermediates, uncovering key architectural modifications in DNA, induced by genotoxic stress or by the associated cellular response. Here, we describe recent modifications of this visualization workflow to implement partial automation of image acquisition and analysis. Coupling this refined workflow with sample preparation procedures that protect R-loop stability allows for direct visualization of R-loop structures on genomic DNA, independently from probes. Combining single-molecule information and DNA content assessment, this approach provides direct estimations of R-loop frequency, size, and burden on genomic DNA.

Keywords:  Automated workflow; DNA content assessment; DNA replication; DNA–RNA hybrid; Direct visualization; Electron microscopy; R-loops; Single-molecule analysis

DOI:  https://doi.org/10.1007/978-1-0716-2477-7_1
Nature. 2022 Jun 15.

Mechanism of replication origin melting nucleated by CMG helicase assembly.

Jacob S Lewis, Marta H Gross, Joana Sousa, Sarah S Henrikus, Julia F Greiwe, Andrea Nans, John F X Diffley, Alessandro Costa.

The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

DOI: https://doi.org/10.1038/s41586-022-04829-4
Mol Oncol. 2022 Jun 16.

Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR-based screens.

Jordan Wilson, Joanna I Loizou.

  The concepts of synthetic lethality and viability have emerged as powerful approaches to identify vulnerabilities and resistances within the DNA damage response for the treatment of cancer. Historically, interactions between two genes have had a longstanding presence in genetics and have been identified through forward genetic screens that rely on the molecular basis of the characterized phenotypes, typically caused by mutations in single genes. While such complex genetic interactions between genes have been studied extensively in model organisms, they have only recently been prioritized as therapeutic strategies due to technological advancements in genetic screens. Here, we discuss synthetic lethal and viable interactions within the DNA damage response and present how CRISPR-based genetic screens and chemical compounds have allowed for the systematic identification and targeting of such interactions for the treatment of cancer.

Keywords:  CRISPR-Cas9 screens; DNA damage response; cancer therapy; drug discovery; synthetic lethality; synthetic viability

DOI:  https://doi.org/10.1002/1878-0261.13272
Front Endocrinol (Lausanne). 2022 ;13 876602

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response.

Camilla Cristalli, Maria Cristina Manara, Sergio Valente, Evelin Pellegrini, Alberto Bavelloni, Alessandra De Feo, William Blalock, Elisabetta Di Bello, David Piñeyro, Angelika Merkel, Manel Esteller, Oscar M Tirado, Antonello Mai, Katia Scotlandi.

  DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS.

Keywords:  DNA damage; DNA methylation; DNMT inhibitors; Drug synergism; PARP inhibitors; doxorubicin; epigenetic therapies; ewing sarcoma

DOI:  https://doi.org/10.3389/fendo.2022.876602
Methods Mol Biol. 2022 ;2528 39-53

Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae.

Juan C Cañas, Andrés Aguilera, Belén Gómez-González.

  R-loops are transcriptional by-products formed by a hybrid of the nascent RNA molecule with its DNA template and the displaced nontemplate DNA strand. The single stranded nature of the displaced nontemplate strand makes it vulnerable to attack. This property is used in nature to cause directed mutagenesis and breaks by the action of the activation-induced cytosine deaminase (AID) enzyme and can thus be exploited to detect the presence of R-loops even when they form at low frequencies by overexpressing this enzyme in vivo or by in vitro treatment with the bisulfite anion, which further allows nucleotide resolution. This is of particular relevance given the fact that R-loops have the potential to hamper DNA replication and repair, threatening genome integrity. Here, we describe the protocols used in the yeast Saccharomyces cerevisiae to infer the presence of R-loops through increased AID-induced DNA damage, measured as increased recombination or Rad52 foci formation as well as to detect single R-loop molecules and determine their length at particular genomic sites via bisulfite treatment and amplification.

Keywords:  AID; Bisulfite; Cytosine deamination; DNA-RNA hybrid; R-loop

DOI:  https://doi.org/10.1007/978-1-0716-2477-7_4
PLoS Genet. 2022 Jun 13. 18(6): e1010275

Site-specific MCM sumoylation prevents genome rearrangements by controlling origin-bound MCM.

Yun Quan, Qian-Yi Zhang, Ann L Zhou, Yuhao Wang, Jiaxi Cai, Yong-Qi Gao, Huilin Zhou.

Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.

DOI: https://doi.org/10.1371/journal.pgen.1010275
Breast Cancer Res. 2022 Jun 17. 24(1): 41

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer.

Leonie Ratz, Chiara Brambillasca, Leandra Bartke, Maxim A Huetzen, Jonas Goergens, Orsolya Leidecker, Ron D Jachimowicz, Marieke van de Ven, Natalie Proost, Bjørn Siteur, Renske de Korte-Grimmerink, Peter Bouwman, Emilia M Pulver, Roebi de Bruijn, Jörg Isensee, Tim Hucho, Gaurav Pandey, Maarten van Lohuizen, Peter Mallmann, Hans Christian Reinhardt, Jos Jonkers, Julian Puppe.

   BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype.
METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors.
RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors.
CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

Keywords:  BRCA1 mutation; Breast cancer; EZH2; Synthetic lethality

DOI:  https://doi.org/10.1186/s13058-022-01534-y
Front Genet. 2022 ;13 884210

The Pathogenic R3052W BRCA2 Variant Disrupts Homology-Directed Repair by Failing to Localize to the Nucleus.

Judit Jimenez-Sainz, Adam Krysztofiak, Jennifer Garbarino, Faye Rogers, Ryan B Jensen.

  The BRCA2 germline missense variant, R3052W, resides in the DNA binding domain and has been previously classified as a pathogenic allele. In this study, we sought to determine how R3052W alters the cellular functions of BRCA2 in the DNA damage response. The BRCA2 R3052W mutated protein exacerbates genome instability, is unable to rescue homology-directed repair, and fails to complement cell survival following exposure to PARP inhibitors and crosslinking drugs. Surprisingly, despite anticipated defects in DNA binding or RAD51-mediated DNA strand exchange, the BRCA2 R3052W protein mislocalizes to the cytoplasm precluding its ability to perform any DNA repair functions. Rather than acting as a simple loss-of-function mutation, R3052W behaves as a dominant negative allele, likely by sequestering RAD51 in the cytoplasm.

Keywords:  BRCA2; DNA repair; DSS1; R3052W; RAD51; homology-directed repair; nuclear localization

DOI:  https://doi.org/10.3389/fgene.2022.884210
Sci Rep. 2022 Jun 17. 12(1): 10163

Efficient discrimination against RNA-containing primers by human DNA polymerase ε.

Alisa E Lisova, Andrey G Baranovskiy, Lucia M Morstadt, Nigar D Babayeva, Tahir H Tahirov.

DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Polε easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolεCD extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3' end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3' end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPolεCD has low affinity to DNA (KD of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPolεCD/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPolε discriminative against RNA and chimeric RNA-DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2'OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.

DOI: https://doi.org/10.1038/s41598-022-14602-2
mSphere. 2022 Jun 13. e0015622

Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania.

Wen-Wei Zhang, Douglas G Wright, Lynn Harrison, Greg Matlashewski.

  In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins) and DNA ligase IV are the core NHEJ factors. Ku could also be involved in other cellular processes, including telomere length regulation, DNA replication, transcription, and translation control. Leishmania, an early branching eukaryote and the causative agent of leishmaniasis, has no functional NHEJ pathway due to its lack of DNA ligase IV and other NHEJ factors but retains Ku70 and Ku80 proteins. In this study, we generated Leishmania donovani Ku70 disruption mutants and Ku70 and Ku80 double gene (Ku70/80) disruption mutants. We found that Leishmania Ku is still involved in DSB repair, possibly through its binding to DNA ends to block and slowdown 5' end resections and Ku-Ku or other protein interactions. Depending on location of a DSB between the direct repeat genomic sequences, Leishmania Ku could have an inhibiting effect, no effect or a promoting effect on the DSB repair mediated by single strand annealing (SSA), the most frequently used DSB repair pathway in Leishmania. Ku70/80 proteins are also required for the healthy proliferation of Leishmania cells. Interestingly, unlike in Trypanosoma brucei and L. mexicana, Ku70/80 proteins are dispensable for maintaining the normal lengths of telomeres in L. donovani. We also show it is possible to reconstitute the two components (Ku and Ligase D) NHEJ pathway derived from Mycobacterium marinum in Leishmania. This improved DSB repair fidelity and efficiency in Leishmania and sets up an example that the bacterial NHEJ pathway can be successfully reconstructed in an NHEJ-deficient eukaryotic parasite. IMPORTANCE Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite Leishmania has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins. In this study, we found that Leishmania Ku heterodimers are still participating in DSB repair possibly through blocking 5' end resections and Ku-Ku protein interactions. Depending on the DSB location, Ku could have an inhibiting or promoting effect on DSB repair mediated by the single-strand annealing repair pathway. Ku is also required for the normal growth of the parasite but surprisingly dispensable for maintaining the telomere lengths. Further, we show it is possible to introduce Mycobacterium marinum NHEJ pathway into Leishmania. Understanding DSB repair mechanisms of Leishmania may improve the CRISPR gene targeting specificity and efficiency and help identify new drug targets for this important human parasite.

Keywords:  CRISPR-Cas9; DSB repair; Ku; Ku70; Ku80; Leishmania donovani; Ligase D; Mycobacterium marinum; NHEJ; SSA; double-strand DNA break; gene deletion; gene targeting; nonhomologous end joining; single-strand annealing; telomere maintenance

DOI:  https://doi.org/10.1128/msphere.00156-22
EMBO Mol Med. 2022 Jun 13. e16171

Targeting the Leukemic stem cell protein machinery by inhibition of mitochondrial pyrimidine synthesis.

Elisa Donato, Andreas Trumpp.

Acute Myeloid Leukemia is one of the most aggressive blood cancers with a high frequency of relapse. While standard chemotherapy is able to target rapidly proliferating immature blasts, it fails to eradicate slowly proliferating Leukemic Stem Cells. Therefore, new therapeutic strategies that efficiently target LSCs are urgently needed. Recent studies suggest that LSCs have particular metabolic vulnerabilities, which would open the possibility of a therapeutic window with limited off-target effects on the normal hematopoietic system. In this issue of EMBO Molecular Medicine, So and colleagues investigate the mechanism of action of AG636, a new potent inhibitor of de novo pyrimidine synthesis, and discovered an unexpected link to AML protein translation essential for LSC function.

DOI: https://doi.org/10.15252/emmm.202216171
Methods Mol Biol. 2022 ;2528 289-303

Proximity Ligation Assay for Detection of R-Loop Complexes upon DNA Damage.

Adele Alagia, Ruth F Ketley, Monika Gullerova.

  In situ Proximity Ligation Assay (PLA ) can be used to detect the close proximity (less than ~40 nm) of two biological molecules of interest in cells. Here we report the application of this method for the specific detection of R-loop interacting proteins and RNA modifications in close proximity to R-loops in non-damage and ionizing radiation (IR) induced DNA damage conditions.

Keywords:  DNA Damage; Double Strand breaks; Proximity Ligation Assay; R-loops; RNA modification; RNA:DNA hybrids

DOI:  https://doi.org/10.1007/978-1-0716-2477-7_19
Mutat Res Rev Mutat Res. 2022 Jan-Jun;789:pii: S1383-5742(21)00037-5. [Epub ahead of print]789 108400

XPC multifaceted roles beyond DNA damage repair: p53-dependent and p53-independent functions of XPC in cell fate decisions.

Abir Zebian, Maya El-Dor, Abdullah Shaito, Frédéric Mazurier, Hamid Reza Rezvani, Kazem Zibara.

  Xeroderma pigmentosum group C protein (XPC) acts as a DNA damage recognition factor for bulky adducts and as an initiator of global genome nucleotide excision repair (GG-NER). Novel insights have shown that the role of XPC is not limited to NER, but is also implicated in DNA damage response (DDR), as well as in cell fate decisions upon stress. Moreover, XPC has a proteolytic role through its interaction with p53 and casp-2S. XPC is also able to determine cellular outcomes through its interaction with downstream proteins, such as p21, ARF, and p16. XPC interactions with effector proteins may drive cells to various fates such as apoptosis, senescence, or tumorigenesis. In this review, we explore XPC's involvement in different molecular pathways in the cell and suggest that XPC can be considered not only as a genomic caretaker and gatekeeper but also as a tumor suppressor and cellular-fate decision maker. These findings envisage that resistance to cell death, induced by DNA-damaging therapeutics, in highly prevalent P53-deficent tumors might be overcome through new therapeutic approaches that aim to activate XPC in these tumors. Moreover, this review encourages care providers to consider XPC status in cancer patients before chemotherapy in order to improve the chances of successful treatment and enhance patients' survival.

Keywords:  Cell fate; DNA damage response; DNA repair; Tumor suppressor; XPC; p53

DOI:  https://doi.org/10.1016/j.mrrev.2021.108400
Trends Immunol. 2022 Jun 11. pii: S1471-4906(22)00116-8. [Epub ahead of print]

Mutagenic repair during antibody diversification: emerging insights.

Yuqing Feng, Alberto Martin.

Deoxyuracils (dUs) produced by activation-induced cytidine deaminase (AID) during antibody diversification are processed by base excision repair (BER) and mismatch repair (MMR) pathways that paradoxically expand this lesion within jawed vertebrate immunoglobulin (Ig) genes. We highlight new findings describing mechanisms that allow B cells to carry out mutagenic DNA repair, an essential process for antibody maturation with implications in cancer pathogenesis.

DOI: https://doi.org/10.1016/j.it.2022.05.004
Am J Cancer Res. 2022 ;12(5): 2293-2309

Chk1 inhibitor-induced DNA damage increases BFL1 and decreases BIM but does not protect human cancer cell lines from Chk1 inhibitor-induced apoptosis.

Andrew J Massey.

V158411 is a potent, selective Chk1 inhibitor currently in pre-clinical development. We utilised RNA-sequencing to evaluate the gene responses to V158411 treatment. BCL2A1 was highly upregulated in U2OS cells in response to V158411 treatment with BCL2A1 mRNA increased > 400-fold in U2OS but not HT29 cells. Inhibitors of Chk1, Wee1 and topoisomerases but not other DNA damaging agents or inhibitors of ATR, ATM or DNA-PKcs increased BFL1 and decreased BIM protein. Increased BFL1 appeared limited to a subset of approximately 35% of U2OS cells. Out of 24 cell lines studied, U2OS cells were unique in being the only cell line with low basal BFL1 levels to be increased in response to DNA damage. Induction of BFL1 in U2OS cells appeared dependent on PI3K/AKT/mTOR/MEK pathway signalling but independent of NF-κB transcription factors. Inhibitors of MEK, mTOR and PI3K effectively blocked the increase in BFL1 following V15841 treatment. Increased BFL1 expression did not block apoptosis in U2OS cells in response to V158411 treatment and cells with high basal expression of BFL1 readily underwent caspase-dependent apoptosis following Chk1 inhibitor therapy. BFL1 induction in response to Chk1 inhibition appeared to be a rare event that was dependent on MEK/PI3K/AKT/mTOR signalling.

Keywords: BFL1; Chk1; DNA damage; MEK; kinase inhibitor
Basic Clin Pharmacol Toxicol. 2022 Jun 16.

Targeting the DNA repair enzymes MTH1 and OGG1 as a novel approach to treat inflammatory diseases.

Stella Karsten.

  Autoimmune diseases and acute inflammation like sepsis cause significant morbidity and disability globally, and new targeted therapies are urgently needed. DNA repair and reactive oxygen species (ROS) pathways have long been investigated as targets for cancer treatment, but their role in immunological research has been limited. In this MiniReview, we discuss the DNA repair enzymes MTH1 and OGG1 as targets to treat both T cell-driven diseases and acute inflammation. The MiniReview is based on a PhD thesis where both enzymes were investigated with cellular and animal models. For MTH1, we found that its inhibition selectively kills activated T cells without being toxic to resting cells or other tissues. MTH1 inhibition also had an alleviating role in disease models of psoriasis and multiple sclerosis. We further identified a novel MTH1low ROSlow phenotype among activated T cells. Regarding OGG1, we demonstrated a mechanism of action of the OGG1 inhibitor TH5487, which prevents the assembly of pro-inflammatory transcription factors and mitigates acute airway infection in mouse models of pneumonia. Hence, we propose both enzymes to be promising novel targets to treat inflammation and suggest that redox- and DNA repair pathways could be useful targets for future immunomodulating therapies.

Keywords:  DNA repair; Drug discovery; Immunology; Reactive Oxygen Species; T cells

DOI:  https://doi.org/10.1111/bcpt.13765
ChemMedChem. 2022 Jun 17.

The first structure of human MTHFD2L and its implications for the development of isoform-selective inhibitors.

Emma Scaletti, Robert Gustafsson Westergren, Yasmin Andersson, Elisee Wiita, Martin Henriksson, Evert J Homan, Ann-Sofie Jemth, Thomas Helleday, Pål Stenmark.

  Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a mitochondrial 1-carbon metabolism enzyme, which is an attractive anticancer drug target as it is highly upregulated in cancer but is not expressed in healthy adult cells. Selective MTHFD2 inhibitors could therefore offer reduced side-effects during treatment, which are common with antifolate drugs that target other 1C-metabolism enzymes. This task is challenging however, as MTHFD2 shares high sequence identity with the constitutively expressed isozymes cytosolic MTHFD1 and mitochondrial MTHFD2L. In fact, one of the most potent MTHFD2 inhibitors reported to date, TH7299, is actually more active against MTHFD1 and MTHFD2L. While structures of MTHFD2 and MTHFD1 exist, no MTHFD2L structures are available. We determined the first structure of MTHFD2L and its complex with TH7299, which reveals the structural basis for its highly potent MTHFD2L inhibition. Detailed analysis of the MTHFD2L structure presented here clearly highlights the challenges associated with developing truly isoform-selective MTHFD2 inhibitors.

Keywords:  1C-metabolism; cancer; enzyme inhibition; methylene tetrahydrofolate dehydrogenase; structural biology

DOI:  https://doi.org/10.1002/cmdc.202200274
Nat Commun. 2022 Jun 17. 13(1): 3493

ZFP281-BRCA2 prevents R-loop accumulation during DNA replication.

Yan Wang, Binbin Ma, Xiaoxu Liu, Ge Gao, Zhuanzhuan Che, Menghan Fan, Siyan Meng, Xiru Zhao, Rio Sugimura, Hua Cao, Zhongjun Zhou, Jing Xie, Chengqi Lin, Zhuojuan Luo.

R-loops are prevalent in mammalian genomes and involved in many fundamental cellular processes. Depletion of BRCA2 leads to aberrant R-loop accumulation, contributing to genome instability. Here, we show that ZFP281 cooperates with BRCA2 in preventing R-loop accumulation to facilitate DNA replication in embryonic stem cells. ZFP281 depletion reduces PCNA levels on chromatin and impairs DNA replication. Mechanistically, we demonstrate that ZFP281 can interact with BRCA2, and that BRCA2 is enriched at G/C-rich promoters and requires both ZFP281 and PRC2 for its proper recruitment to the bivalent chromatin at the genome-wide scale. Furthermore, depletion of ZFP281 or BRCA2 leads to accumulation of R-loops over the bivalent regions, and compromises activation of the developmental genes by retinoic acid during stem cell differentiation. In summary, our results reveal that ZFP281 recruits BRCA2 to the bivalent chromatin regions to ensure proper progression of DNA replication through preventing persistent R-loops.

DOI: https://doi.org/10.1038/s41467-022-31211-9
Cureus. 2022 May;14(5): e24976

Remarkable Response of Hairy Cell Leukemia Variant to Single-Agent Cladribine.

Steve B Otieno, Osarenren Ogbeide.

  Classical hairy cell leukemia (cHCL) and related mature lymphoid B-cell neoplasms including hairy cell leukemia variant (HCLv) and splenic diffuse red pulp lymphoma (SDRPL) are a rare subset of lymphoid neoplasms. cHCL accounts for around 2% of all leukemias and is characterized by a peripheral smear with large lymphoid cells with cytoplasmic projections giving the cells a hairy appearance, splenomegaly, and cytopenias. Majority of cHCL cases harbor a BRAFV600E mutation. cHCL usually responds well to single-agent purine analogs. HCLv is even rarer and constitutes around 0.4% of lymphoid malignancies. Unlike cHCL, HCLv is less responsive to standard single-agent purine analogs and typically does not harbor the BRAFV600E mutation. The "hairy cells," splenomegaly, and cytopenias are common in both. We report a case of a patient with HCLv who was treated with a single purine analog and achieved a near-complete response.

Keywords:  brafv600e; cytopenias; hairy cell leukemia variant; purine analog; splenomegaly

DOI:  https://doi.org/10.7759/cureus.24976
Nat Rev Drug Discov. 2022 Jun 16.

What's next for the synthetic lethality drug discovery engine?

Asher Mullard.

DOI: https://doi.org/10.1038/d41573-022-00107-0
ACS Chem Biol. 2022 Jun 14.

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo.

Verena N Schreier, Morten O Loehr, Evelyn Lattmann, Nathan W Luedtke.

Modified nucleoside triphosphates (NTPs) are powerful probes and medicines, but their anionic character impedes membrane permeability. As such, invasive delivery techniques, transport carriers, or prodrug strategies are required for their in vivo use. Here, we present a fluorescent 2'-deoxyribonucleoside triphosphate "TAMRA-dATP" that exhibits surprisingly high bioavailability in vivo. TAMRA-dATP spontaneously forms nanoparticles in Mg+2-containing buffers that are taken into the vesicles of living cells and animals by energy-dependent processes. In cell cultures, photochemical activation with yellow laser light (561 nm) facilitated endosomal escape of TAMRA-dATP, resulting in its metabolic incorporation into DNA in vitro. In contrast, in vivo studies revealed that TAMRA-dATP is extensively trafficked by active pathways into cellular DNA of zebrafish (Danio rerio) and Caenorhabditis elegans where DNA labeling was observed in live animals, even without photochemical release. Metabolic labeling of DNA in whole, living animals can therefore be achieved by simply soaking animals in a buffer containing TAMRA-dATP or a structurally related compound, Cy3-dATP.

DOI: https://doi.org/10.1021/acschembio.2c00153
Nature. 2022 Jun 15.

A pan-cancer compendium of chromosomal instability.

Ruben M Drews, Barbara Hernando, Maxime Tarabichi, Kerstin Haase, Tom Lesluyes, Philip S Smith, Lena Morrill Gavarró, Dominique-Laurent Couturier, Lydia Liu, Michael Schneider, James D Brenton, Peter Van Loo, Geoff Macintyre, Florian Markowetz.

Chromosomal instability (CIN) results in the accumulation of large-scale losses, gains and rearrangements of DNA1. The broad genomic complexity caused by CIN is a hallmark of cancer2; however, there is no systematic framework to measure different types of CIN and their effect on clinical phenotypes pan-cancer. Here we evaluate the extent, diversity and origin of CIN across 7,880 tumours representing 33 cancer types. We present a compendium of 17 copy number signatures that characterize specific types of CIN, with putative aetiologies supported by multiple independent data sources. The signatures predict drug response and identify new drug targets. Our framework refines the understanding of impaired homologous recombination, which is one of the most therapeutically targetable types of CIN. Our results illuminate a fundamental structure underlying genomic complexity in human cancers and provide a resource to guide future CIN research.

DOI: https://doi.org/10.1038/s41586-022-04789-9