bims-numges 2021-11-28 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

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

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair.
FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.
Fam72a enforces error-prone DNA repair during antibody diversification.
Adaptation to Chronic-Cycling Hypoxia Renders Cancer Cells Resistant to MTH1-Inhibitor Treatment Which Can Be Counteracted by Glutathione Depletion.
DNA End Resection: Mechanism and Control.
Alternative Lengthening of Telomeres: Lessons to Be Learned from Telomeric DNA Double-Strand Break Repair.
Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation.
Impact of G-Quadruplexes and Chronic Inflammation on Genome Instability: Additive Effects during Carcinogenesis.
Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.
Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.
Repurposing ceritinib induces DNA damage and enhances PARP inhibitor responses in high-grade serous ovarian carcinoma.
Sir3 heterochromatin protein promotes non-homologous end joining by direct inhibition of Sae2.
SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.
NAD+ bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.
Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation.
Identification of a Chemotherapeutic Lead Molecule for the Potential Disruption of the FAM72A-UNG2 Interaction to Interfere with Genome Stability, Centromere Formation, and Genome Editing.
Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.
hENT1 Predicts Benefit from Gemcitabine in Pancreatic Cancer but Only with Low CDA mRNA.
Searching for DNA Damage: Insights From Single Molecule Analysis.
Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms.
ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.
Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.
Transcriptional and Metabolic Investigation in 5'-Nucleotidase Deficient Cancer Cell Lines.
The role of chromatin at transcription-replication conflicts as a genome safeguard.
Synthesis, characterization and biological evaluation of thiadiazole amide derivatives as nucleoside triphosphate diphosphohydrolases (NTPDases) inhibitors.
PCNA Loaders and Unloaders-One Ring That Rules Them All.
A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.
Exploring the Structures and Functions of Macromolecular SLX4-Nuclease Complexes in Genome Stability.
Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in Adult T-cell leukemia/lymphoma.
The Relevance of G-Quadruplexes for DNA Repair.
Targeting the DNA damage response: PARP inhibitors and new perspectives in the landscape of cancer treatment.
Genomic Instability and Cancer Risk Associated with Erroneous DNA Repair.
Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology.
The cardiomyocyte disrupts pyrimidine biosynthesis in non-myocytes to regulate heart repair.
Genomic instability, inflammatory signaling and response to cancer immunotherapy.
Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination.
Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response.
Role of PARP in TNBC: Mechanism of Inhibition, Clinical Applications, and Resistance.
Effects of 5',8'-Cyclo-2'-Deoxypurines on the Base Excision Repair of Clustered DNA Lesions in Nuclear Extracts of the XPC Cell Line.

Nat Cell Biol. 2021 Nov 22.

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair.

Marek Adamowicz, Richard Hailstone, Annie A Demin, Emilia Komulainen, Hana Hanzlikova, Jan Brazina, Amit Gautam, Sophie E Wells, Keith W Caldecott.

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

DOI: https://doi.org/10.1038/s41556-021-00792-w
Nature. 2021 Nov 24.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Yuqing Feng, Conglei Li, Jessica A Stewart, Philip Barbulescu, Noé Seija Desivo, Alejandro Álvarez-Quilón, Rossanna C Pezo, Madusha L W Perera, Katherine Chan, Amy Hin Yan Tong, Rukshana Mohamad-Ramshan, Maribel Berru, Diana Nakib, Gavin Li, Gholam Ali Kardar, James R Carlyle, Jason Moffat, Daniel Durocher, Javier M Di Noia, Ashok S Bhagwat, Alberto Martin.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

DOI: https://doi.org/10.1038/s41586-021-04144-4
Nature. 2021 Nov 24.

Fam72a enforces error-prone DNA repair during antibody diversification.

Mélanie Rogier, Jacques Moritz, Isabelle Robert, Chloé Lescale, Vincent Heyer, Arthur Abello, Ophélie Martin, Katia Capitani, Morgane Thomas, Anne-Sophie Thomas-Claudepierre, Brice Laffleur, Florence Jouan, Eric Pinaud, Karin Tarte, Michel Cogné, Silvestro G Conticello, Evi Soutoglou, Ludovic Deriano, Bernardo Reina-San-Martin.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

DOI: https://doi.org/10.1038/s41586-021-04093-y
Cells. 2021 Nov 05. pii: 3040. [Epub ahead of print]10(11):

Adaptation to Chronic-Cycling Hypoxia Renders Cancer Cells Resistant to MTH1-Inhibitor Treatment Which Can Be Counteracted by Glutathione Depletion.

Christine Hansel, Julian Hlouschek, Kexu Xiang, Margarita Melnikova, Juergen Thomale, Thomas Helleday, Verena Jendrossek, Johann Matschke.

  Tumor hypoxia and hypoxic adaptation of cancer cells represent major barriers to successful cancer treatment. We revealed that improved antioxidant capacity contributes to increased radioresistance of cancer cells with tolerance to chronic-cycling severe hypoxia/reoxygenation stress. We hypothesized, that the improved tolerance to oxidative stress will increase the ability of cancer cells to cope with ROS-induced damage to free deoxy-nucleotides (dNTPs) required for DNA replication and may thus contribute to acquired resistance of cancer cells in advanced tumors to antineoplastic agents inhibiting the nucleotide-sanitizing enzyme MutT Homologue-1 (MTH1), ionizing radiation (IR) or both. Therefore, we aimed to explore potential differences in the sensitivity of cancer cells exposed to acute and chronic-cycling hypoxia/reoxygenation stress to the clinically relevant MTH1-inhibitor TH1579 (Karonudib) and to test whether a multi-targeting approach combining the glutathione withdrawer piperlongumine (PLN) and TH1579 may be suited to increase cancer cell sensitivity to TH1579 alone and in combination with IR. Combination of TH1579 treatment with radiotherapy (RT) led to radiosensitization but was not able to counteract increased radioresistance induced by adaptation to chronic-cycling hypoxia/reoxygenation stress. Disruption of redox homeostasis using PLN sensitized anoxia-tolerant cancer cells to MTH1 inhibition by TH1579 under both normoxic and acute hypoxic treatment conditions. Thus, we uncover a glutathione-driven compensatory resistance mechanism towards MTH1-inhibition in form of increased antioxidant capacity as a consequence of microenvironmental or therapeutic stress.

Keywords:  Karonudib; TH1579; antioxidant capacity; chronic cycling hypoxia; ionizing radiation; metabolic reprogramming; piperlongumine; radiation resistance; redox homeostasis

DOI:  https://doi.org/10.3390/cells10113040
Annu Rev Genet. 2021 Nov 23. 55 285-307

DNA End Resection: Mechanism and Control.

Petr Cejka, Lorraine S Symington.

  DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'→3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.

Keywords:  53BP1; CtIP; DNA repair; DNA2; EXO1; MRE11-RAD50-NBS1; homologous recombination

DOI:  https://doi.org/10.1146/annurev-genet-071719-020312
Genes (Basel). 2021 Oct 29. pii: 1734. [Epub ahead of print]12(11):

Alternative Lengthening of Telomeres: Lessons to Be Learned from Telomeric DNA Double-Strand Break Repair.

Thomas Kent, David Clynes.

  The study of the molecular pathways underlying cancer has given us important insights into how breaks in our DNA are repaired and the dire consequences that can occur when these processes are perturbed. Extensive research over the past 20 years has shown that the key molecular event underpinning a subset of cancers involves the deregulated repair of DNA double-strand breaks (DSBs) at telomeres, which in turn leads to telomere lengthening and the potential for replicative immortality. Here we discuss, in-depth, recent major breakthroughs in our understanding of the mechanisms underpinning this pathway known as the alternative lengthening of telomeres (ALT). We explore how this gives us important insights into how DSB repair at telomeres is regulated, with relevance to the cell-cycle-dependent regulation of repair, repair of stalled replication forks and the spatial regulation of DSB repair.

Keywords:  ATRX; alternative lengthening of telomeres; break-induced replication; double-strand break repair; telomeres

DOI:  https://doi.org/10.3390/genes12111734
iScience. 2021 Nov 19. 24(11): 103354

Fumarase affects the deoxyribonucleic acid damage response by protecting the mitochondrial desulfurase Nfs1p from modification and inactivation.

Joyce Yip, Suqing Wang, Jasper Tan, Teck Kwang Lim, Qingsong Lin, Zhang Yu, Ofri Karmon, Ophry Pines, Norbert Lehming.

  The Krebs cycle enzyme fumarase, which has been identified as a tumor suppressor, is involved in the deoxyribonucleic acid (DNA) damage response (DDR) in human, yeast, and bacterial cells. We have found that the overexpression of the cysteine desulfurase Nfs1p restores DNA repair in fumarase-deficient yeast cells. Nfs1p accumulates inactivating post-translational modifications in yeast cells lacking fumarase under conditions of DNA damage. Our model is that in addition to metabolic signaling of the DDR in the nucleus, fumarase affects the DDR by protecting the desulfurase Nfs1p in mitochondria from modification and inactivation. Fumarase performs this protection by directly binding to Nfs1p in mitochondria and enabling, the maintenance, via metabolism, of a non-oxidizing environment in mitochondria. Nfs1p is required for the formation of Fe-S clusters, which are essential cofactors for DNA repair enzymes. Thus, we propose that the overexpression of Nfs1p overcomes the lack of fumarase by enhancing the activity of DNA repair enzymes.

Keywords:  Biochemistry; Biological sciences; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2021.103354
Genes (Basel). 2021 Nov 09. pii: 1779. [Epub ahead of print]12(11):

Impact of G-Quadruplexes and Chronic Inflammation on Genome Instability: Additive Effects during Carcinogenesis.

MaryElizabeth Stein, Kristin A Eckert.

  Genome instability is an enabling characteristic of cancer, essential for cancer cell evolution. Hotspots of genome instability, from small-scale point mutations to large-scale structural variants, are associated with sequences that potentially form non-B DNA structures. G-quadruplex (G4) forming motifs are enriched at structural variant endpoints in cancer genomes. Chronic inflammation is a physiological state underlying cancer development, and oxidative DNA damage is commonly invoked to explain how inflammation promotes genome instability. We summarize where G4s and oxidative stress overlap, with a focus on DNA replication. Guanine has low ionization potential, making G4s vulnerable to oxidative damage. Impacts to G4 structure are dependent upon lesion type, location, and G4 conformation. Occasionally, G4s pose a challenge to replicative DNA polymerases, requiring specialized DNA polymerases to maintain genome stability. Therefore, chronic inflammation creates a dual challenge for DNA polymerases to maintain genome stability: faithful G4 synthesis and bypassing unrepaired oxidative lesions. Inflammation is also accompanied by global transcriptome changes that may impact mutagenesis. Several studies suggest a regulatory role for G4s within cancer- and inflammatory-related gene promoters. We discuss the extent to which inflammation could influence gene regulation by G4s, thereby impacting genome instability, and highlight key areas for new investigation.

Keywords:  DNA polymerases; G-quadruplex; genome instability; oxidative stress

DOI:  https://doi.org/10.3390/genes12111779
DNA Repair (Amst). 2021 Nov 17. pii: S1568-7864(21)00203-2. [Epub ahead of print]109 103247

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Carolyn G Marsden, Lipsa Das, Timothy P Nottoli, Scott D Kathe, Sylvie Doublié, Susan S Wallace, Joann B Sweasy.

  Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Keywords:  Base excision repair; Genomic instability; NTHL1 glycosylase; Oxidative DNA damage

DOI:  https://doi.org/10.1016/j.dnarep.2021.103247
Nucleic Acids Res. 2021 Nov 19. pii: gkab1068. [Epub ahead of print]

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.

Yoko Hayashi-Takanaka, Yuichiro Hayashi, Yasuhiro Hirano, Atsuko Miyawaki-Kuwakado, Yasuyuki Ohkawa, Chikashi Obuse, Hiroshi Kimura, Tokuko Haraguchi, Yasushi Hiraoka.

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.

DOI: https://doi.org/10.1093/nar/gkab1068
Cancer Res. 2021 Nov 22. pii: canres.CAN-21-0732-A.2021. [Epub ahead of print]

Repurposing ceritinib induces DNA damage and enhances PARP inhibitor responses in high-grade serous ovarian carcinoma.

Arun Kanakkanthara, Xiaonan Hou, Thomas L Ekstrom, Valentina Zanfagnin, Amelia M Huehls, Rebecca L Kelly, Husheng Ding, Melissa C Larson, George Vasmatzis, Ann L Oberg, Scott H Kaufmann, Aaron S Mansfield, S John Weroha, Larry M Karnitz.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. Additionally, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0732
EMBO J. 2021 Nov 24. e108813

Sir3 heterochromatin protein promotes non-homologous end joining by direct inhibition of Sae2.

Hélène Bordelet, Rafaël Costa, Clémentine Brocas, Jordane Dépagne, Xavier Veaute, Didier Busso, Amandine Batté, Raphaël Guérois, Stéphane Marcand, Karine Dubrana.

  Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin-like chromatin at sub-telomeric chromosomal regions. SIR-mediated repressive chromatin limits DNA double-strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR-mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2-dependent functions of the MRX (Mre11-Rad50-Xrs2) complex, thereby limiting Mre11-mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ.

Keywords:  DSB repair; NHEJ; Sae2; Sir3; heterochromatin

DOI:  https://doi.org/10.15252/embj.2021108813
Cell Rep. 2021 Nov 23. pii: S2211-1247(21)01516-3. [Epub ahead of print]37(8): 110034

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Nataliia Serbyn, Ivona Bagdiul, Audrey Noireterre, Agnès H Michel, Raymond T Suhandynata, Huilin Zhou, Benoît Kornmann, Françoise Stutz.

  Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

Keywords:  DNA-protein crosslink; DPC repair; Flp-nick; STUbL; SUMO; Siz2; Tdp1; Top1; Top1cc; Ulp1; Wss1

DOI:  https://doi.org/10.1016/j.celrep.2021.110034
NAR Cancer. 2021 Dec;3(4): zcab044

NAD+ bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.

Jianfeng Li, Kate M Saville, Md Ibrahim, Xuemei Zeng, Steve McClellan, Anusha Angajala, Alison Beiser, Joel F Andrews, Mai Sun, Christopher A Koczor, Jennifer Clark, Faisal Hayat, Mikhail V Makarov, Anna Wilk, Nathan A Yates, Marie E Migaud, Robert W Sobol.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

DOI: https://doi.org/10.1093/narcan/zcab044
J Biomed Sci. 2021 Nov 24. 28(1): 81

Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation.

Wei-Ting Chen, Huan-Yi Tseng, Chung-Lin Jiang, Chih-Ying Lee, Peter Chi, Liuh-Yow Chen, Kai-Yin Lo, I-Ching Wang, Fu-Jung Lin.

   BACKGROUND: RAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.
METHODS: To investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach.
RESULTS: Here, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion.
CONCLUSIONS: Our study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Keywords:  DNA damage; Elp1; Homologous recombination; RAD51; Translational regulation

DOI:  https://doi.org/10.1186/s12929-021-00773-z
Cancers (Basel). 2021 Nov 22. pii: 5870. [Epub ahead of print]13(22):

Identification of a Chemotherapeutic Lead Molecule for the Potential Disruption of the FAM72A-UNG2 Interaction to Interfere with Genome Stability, Centromere Formation, and Genome Editing.

Senthil Renganathan, Subrata Pramanik, Rajasekaran Ekambaram, Arne Kutzner, Pok-Son Kim, Klaus Heese.

  Family with sequence similarity 72 A (FAM72A) is a pivotal mitosis-promoting factor that is highly expressed in various types of cancer. FAM72A interacts with the uracil-DNA glycosylase UNG2 to prevent mutagenesis by eliminating uracil from DNA molecules through cleaving the N-glycosylic bond and initiating the base excision repair pathway, thus maintaining genome integrity. In the present study, we determined a specific FAM72A-UNG2 heterodimer protein interaction using molecular docking and dynamics. In addition, through in silico screening, we identified withaferin B as a molecule that can specifically prevent the FAM72A-UNG2 interaction by blocking its cell signaling pathways. Our results provide an excellent basis for possible therapeutic approaches in the clinical treatment of cancer.

Keywords:  DNA repair; cell cycle; centromere; proliferation

DOI:  https://doi.org/10.3390/cancers13225870
Proc Natl Acad Sci U S A. 2021 Nov 30. pii: e2106477118. [Epub ahead of print]118(48):

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.

Shyam Twayana, Albino Bacolla, Angelica Barreto-Galvez, Ruth B De-Paula, William C Drosopoulos, Settapong T Kosiyatrakul, Eric E Bouhassira, John A Tainer, Advaitha Madireddy, Carl L Schildkraut.

  Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

Keywords:  SNP; common fragile sites; non-B DNA; polymerase eta; replication fork pause

DOI:  https://doi.org/10.1073/pnas.2106477118
Cancers (Basel). 2021 Nov 17. pii: 5758. [Epub ahead of print]13(22):

hENT1 Predicts Benefit from Gemcitabine in Pancreatic Cancer but Only with Low CDA mRNA.

Karen Aughton, Nils O Elander, Anthony Evans, Richard Jackson, Fiona Campbell, Eithne Costello, Christopher M Halloran, John R Mackey, Andrew G Scarfe, Juan W Valle, Ross Carter, David Cunningham, Niall C Tebbutt, David Goldstein, Jennifer Shannon, Bengt Glimelius, Thilo Hackert, Richard M Charnley, Alan Anthoney, Markus M Lerch, Julia Mayerle, Daniel H Palmer, Markus W Büchler, Paula Ghaneh, John P Neoptolemos, William Greenhalf.

  Gemcitabine or 5-fluorouracil (5-FU) based treatments can be selected for pancreatic cancer. Equilibrative nucleoside transporter 1 (hENT1) predicts adjuvant gemcitabine treatment benefit over 5-FU. Cytidine deaminase (CDA), inside or outside of the cancer cell, will deaminate gemcitabine, altering transporter affinity. ESPAC-3(v2) was a pancreatic cancer trial comparing adjuvant gemcitabine and 5-FU. Tissue microarray sections underwent in situ hybridization and immunohistochemistry. Analysis of both CDA and hENT1 was possible with 277 patients. The transcript did not correlate with protein levels for either marker. High hENT1 protein was prognostic with gemcitabine; median overall survival was 26.0 v 16.8 months (p = 0.006). Low CDA transcript was prognostic regardless of arm; 24.8 v 21.2 months with gemcitabine (p = 0.02) and 26.4 v 14.6 months with 5-FU (p = 0.02). Patients with low hENT1 protein did better with 5-FU, but only if the CDA transcript was low (median survival of 5-FU v gemcitabine; 29.3 v 18.3 months, compared with 14.2 v 14.6 with high CDA). CDA mRNA is an independent prognostic biomarker. When added to hENT1 protein status, it may also provide treatment-specific predictive information and, within the frame of a personalized treatment strategy, guide to either gemcitabine or 5FU for the individual patient.

Keywords:  5-fluorouracil; biomarker; chemotherapy; gemcitabine; predictive marker; prognostic marker; pyrimidine

DOI:  https://doi.org/10.3390/cancers13225758
Front Mol Biosci. 2021 ;8 772877

Searching for DNA Damage: Insights From Single Molecule Analysis.

Matthew A Schaich, Bennett Van Houten.

  DNA is under constant threat of damage from a variety of chemical and physical insults, such as ultraviolet rays produced by sunlight and reactive oxygen species produced during respiration or inflammation. Because damaged DNA, if not repaired, can lead to mutations or cell death, multiple DNA repair pathways have evolved to maintain genome stability. Two repair pathways, nucleotide excision repair (NER) and base excision repair (BER), must sift through large segments of nondamaged nucleotides to detect and remove rare base modifications. Many BER and NER proteins share a common base-flipping mechanism for the detection of modified bases. However, the exact mechanisms by which these repair proteins detect their damaged substrates in the context of cellular chromatin remains unclear. The latest generation of single-molecule techniques, including the DNA tightrope assay, atomic force microscopy, and real-time imaging in cells, now allows for nearly direct visualization of the damage search and detection processes. This review describes several mechanistic commonalities for damage detection that were discovered with these techniques, including a combination of 3-dimensional and linear diffusion for surveying damaged sites within long stretches of DNA. We also discuss important findings that DNA repair proteins within and between pathways cooperate to detect damage. Finally, future technical developments and single-molecule studies are described which will contribute to the growing mechanistic understanding of DNA damage detection.

Keywords:  DNA damage; DNA tightrope; base excision repair; nucleotide excision repair; single molecule fluorescence microscopy

DOI:  https://doi.org/10.3389/fmolb.2021.772877
Cancer Chemother Pharmacol. 2021 Nov 26.

Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms.

Keishi Yoshida, Atsushi Fujita, Hidehiko Narazaki, Takeshi Asano, Yasuhiko Itoh.

   PURPOSE: Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine.
METHODS: Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation.
RESULTS: The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic.
CONCLUSION: This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms.

Keywords:  Deoxycytidine kinase; Drug resistance; Epigenetics; Leukemia; Nelarabine; Vorinostat

DOI:  https://doi.org/10.1007/s00280-021-04373-4
Nat Commun. 2021 Nov 24. 12(1): 6813

ATR inhibition enables complete tumour regression in ALK-driven NB mouse models.

Joanna Szydzik, Dan E Lind, Badrul Arefin, Yeshwant Kurhe, Ganesh Umapathy, Joachim Tetteh Siaw, Arne Claeys, Jonatan L Gabre, Jimmy Van den Eynden, Bengt Hallberg, Ruth H Palmer.

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

DOI: https://doi.org/10.1038/s41467-021-27057-2
Front Oncol. 2021 ;11 752933

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Donna M Edwards, Dana K Mitchell, Zahi Abdul-Sater, Ka-Kui Chan, Zejin Sun, Aditya Sheth, Ying He, Li Jiang, Jin Yuan, Richa Sharma, Magdalena Czader, Pei-Ju Chin, Yie Liu, Guillermo de Cárcer, Grzegorz Nalepa, Hal E Broxmeyer, D Wade Clapp, Elizabeth A Sierra Potchanant.

  Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

Keywords:  FANCC; Fanconi anemia; genomic instability; leukemia; spindle assembly checkpoint

DOI:  https://doi.org/10.3389/fonc.2021.752933
Cells. 2021 Oct 28. pii: 2918. [Epub ahead of print]10(11):

Transcriptional and Metabolic Investigation in 5'-Nucleotidase Deficient Cancer Cell Lines.

Octavia Cadassou, Prescillia Forey, Christelle Machon, Edoardo Petrotto, Kamel Chettab, Maria Grazia Tozzi, Jérôme Guitton, Charles Dumontet, Emeline Cros-Perrial, Lars Petter Jordheim.

  Enzymes of nucleoside and nucleotide metabolism regulate important cellular processes with potential impacts on nucleotide-unrelated parameters. We have used a set of CRISPR/Cas9-modified cell models expressing both, one, or none of the 5'-nucleotidases cN-II and CD73, together with RNA sequencing and targeted metabolomics, to decipher new regulatory roles of these proteins. We observed important transcriptional modifications between models as well as upon exposure to adenosine. Metabolite content varied differently between cell models in response to adenosine exposure but was rather similar in control conditions. Our original cell models allowed us to identify a new unobvious link between proteins in the nucleotide metabolism and other cellular pathways. Further analyses of our models, including additional experiments, could help us to better understand some of the roles played by these enzymes.

Keywords:  CD73; cN-II; metabolic pathways; transcriptional regulation

DOI:  https://doi.org/10.3390/cells10112918
Biochem Soc Trans. 2021 Nov 25. pii: BST20210691. [Epub ahead of print]

The role of chromatin at transcription-replication conflicts as a genome safeguard.

Aleix Bayona-Feliu, Andrés Aguilera.

  DNA replication ensures the correct copying of the genome and the faithful transfer of the genetic information to the offspring. However, obstacles to replication fork (RF) progression cause RF stalling and compromise efficient genome duplication. Since replication uses the same DNA template as transcription, both transcription and replication must be coordinated to prevent Transcription-Replication Conflicts (TRCs) that could stall RF progression. Several factors contribute to limit the occurrence of such conflicts and their harmful impact on genome integrity. Increasing evidence indicates that chromatin homeostasis plays a key role in the cellular response to TRCs as well as in the preservation of genome integrity. Indeed, chromatin regulating enzymes are frequently mutated in cancer cells, a common characteristic of which is genome instability. Therefore, understanding the role of chromatin in TRC occurrence and resolution may help identify the molecular mechanism by which chromatin protects genome integrity, and the causes and physiological relevance of the high mutation rates of chromatin regulating factors in cancer. Here we review the current knowledge in the field, as well as the perspectives and future applications.

Keywords:  DNA breaks; R-loops; cancer; chromatin; genome instability; transcription-replication conflicts

DOI:  https://doi.org/10.1042/BST20210691
Bioorg Chem. 2021 Nov 14. pii: S0045-2068(21)00833-6. [Epub ahead of print]118 105456

Synthesis, characterization and biological evaluation of thiadiazole amide derivatives as nucleoside triphosphate diphosphohydrolases (NTPDases) inhibitors.

Sadia Abbas, Saira Afzal, Humaira Nadeem, Dilawar Hussain, Peter Langer, Jean Sévigny, Zaman Ashraf, Jamshed Iqbal.

  Importance of extracellular nucleotides is widely understood. These nucleotides act as ligand for P2X and P2Y receptors and modulate a variety of biological functions. However, their extracellular concentration is maintained by a chain of enzymes termed as ecto-nucleotidases. Amongst them, nucleoside triphosphate diphosphohydrolases (NTPDases) is an important enzyme family responsible for the dephosphorylation of these nucleotides. Overexpression of NTPDases leads to many pathological conditions such as cancer and thrombosis. So far, only a few NTPDase inhibitors have been reported. Considering this scarcity of (NTPDase) inhibitors, a number of thiadiazole amide derivatives were synthesized and screened against human (h)-NTPDases. Several compounds showed promising inhibitory activity; compound 5a (IC50 (µM); 0.05 ± 0.008) and 5g (IC50 (µM); 0.04 ± 0.006) appeared to be the most distinguished molecules corresponding to h-NTPDase1 and -2. However, h-NTPDase3 was the least susceptible isozyme and only three compounds (5d, 5e, 5j) strongly inhibited h-NTPDase3. Interestingly, compound 5e was recognized as the most active compound that showed dual inhibition against h-NTPDase3 as well as against h-NTPDase8. For better comprehension of binding mode of these inhibitors, most potent inhibitors were docked with their respective isozyme.

Keywords:  Amide; Ectonucleotidases; Molecular docking; Nucleoside triphosphate diphosphohydrolases; Thiadiazole

DOI:  https://doi.org/10.1016/j.bioorg.2021.105456
Genes (Basel). 2021 Nov 18. pii: 1812. [Epub ahead of print]12(11):

PCNA Loaders and Unloaders-One Ring That Rules Them All.

Matan Arbel, Karan Choudhary, Ofri Tfilin, Martin Kupiec.

  During each cell duplication, the entirety of the genomic DNA in every cell must be accurately and quickly copied. Given the short time available for the chore, the requirement of many proteins, and the daunting amount of DNA present, DNA replication poses a serious challenge to the cell. A high level of coordination between polymerases and other DNA and chromatin-interacting proteins is vital to complete this task. One of the most important proteins for maintaining such coordination is PCNA. PCNA is a multitasking protein that forms a homotrimeric ring that encircles the DNA. It serves as a processivity factor for DNA polymerases and acts as a landing platform for different proteins interacting with DNA and chromatin. Therefore, PCNA is a signaling hub that influences the rate and accuracy of DNA replication, regulates DNA damage repair, controls chromatin formation during the replication, and the proper segregation of the sister chromatids. With so many essential roles, PCNA recruitment and turnover on the chromatin is of utmost importance. Three different, conserved protein complexes are in charge of loading/unloading PCNA onto DNA. Replication factor C (RFC) is the canonical complex in charge of loading PCNA during the S-phase. The Ctf18 and Elg1 (ATAD5 in mammalian) proteins form complexes similar to RFC, with particular functions in the cell's nucleus. Here we summarize our current knowledge about the roles of these important factors in yeast and mammals.

Keywords:  Ctf18; Elg1; PCNA; RFC; SUMO; clamp loading and unloading; ubiquitin

DOI:  https://doi.org/10.3390/genes12111812
J Biol Chem. 2021 Nov 20. pii: S0021-9258(21)01250-3. [Epub ahead of print] 101441

A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.

Whitney M Cleghorn, Anika L Burrell, Michelle M Giarmarco, Daniel C Brock, Yekai Wang, Zachary S Chambers, Jianhai Du, Justin M Kollman, Susan E Brockerhoff.

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene sub-functionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. Loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.

DOI: https://doi.org/10.1016/j.jbc.2021.101441
Front Genet. 2021 ;12 784167

Exploring the Structures and Functions of Macromolecular SLX4-Nuclease Complexes in Genome Stability.

Brandon J Payliss, Ayushi Patel, Anneka C Sheppard, Haley D M Wyatt.

  All organisms depend on the ability of cells to accurately duplicate and segregate DNA into progeny. However, DNA is frequently damaged by factors in the environment and from within cells. One of the most dangerous lesions is a DNA double-strand break. Unrepaired breaks are a major driving force for genome instability. Cells contain sophisticated DNA repair networks to counteract the harmful effects of genotoxic agents, thus safeguarding genome integrity. Homologous recombination is a high-fidelity, template-dependent DNA repair pathway essential for the accurate repair of DNA nicks, gaps and double-strand breaks. Accurate homologous recombination depends on the ability of cells to remove branched DNA structures that form during repair, which is achieved through the opposing actions of helicases and structure-selective endonucleases. This review focuses on a structure-selective endonuclease called SLX1-SLX4 and the macromolecular endonuclease complexes that assemble on the SLX4 scaffold. First, we discuss recent developments that illuminate the structure and biochemical properties of this somewhat atypical structure-selective endonuclease. We then summarize the multifaceted roles that are fulfilled by human SLX1-SLX4 and its associated endonucleases in homologous recombination and genome stability. Finally, we discuss recent work on SLX4-binding proteins that may represent integral components of these macromolecular nuclease complexes, emphasizing the structure and function of a protein called SLX4IP.

Keywords:  MUS81-EME1; SLX1-SLX4; SLX4IP; XPF-ERCC1; genome stability; homologous recombination (HR); structure-selective endonuclease

DOI:  https://doi.org/10.3389/fgene.2021.784167
Blood. 2021 Nov 24. pii: blood.2021012734. [Epub ahead of print]

Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in Adult T-cell leukemia/lymphoma.

Takashi Ishio, Sarvesh Kumar, Joji Shimono, Anusara Daenthanasanmak, Sigrid Dubois, Yuquan Lin, Bonita R Bryant, Michael N Petrus, Emmanuel Bachy, Da Wei Huang, Yandan Yang, Patrick L Green, Hiroo Hasegawa, Michiyuki Maeda, Hideki Goto, Tomoyuki Endo, Takashi Yokota, Kanako C Hatanaka, Yutaka Hatanaka, Shinya Tanaka, Yoshihiro Matsuno, Yibin Yang, Satoshi Hashino, Takanori Teshima, Thomas A Waldmann, Louis M Staudt, Masao Nakagawa.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

DOI: https://doi.org/10.1182/blood.2021012734
Int J Mol Sci. 2021 Nov 22. pii: 12599. [Epub ahead of print]22(22):

The Relevance of G-Quadruplexes for DNA Repair.

Rebecca Linke, Michaela Limmer, Stefan A Juranek, Annkristin Heine, Katrin Paeschke.

  DNA molecules can adopt a variety of alternative structures. Among these structures are G-quadruplex DNA structures (G4s), which support cellular function by affecting transcription, translation, and telomere maintenance. These structures can also induce genome instability by stalling replication, increasing DNA damage, and recombination events. G-quadruplex-driven genome instability is connected to tumorigenesis and other genetic disorders. In recent years, the connection between genome stability, DNA repair and G4 formation was further underlined by the identification of multiple DNA repair proteins and ligands which bind and stabilize said G4 structures to block specific DNA repair pathways. The relevance of G4s for different DNA repair pathways is complex and depends on the repair pathway itself. G4 structures can induce DNA damage and block efficient DNA repair, but they can also support the activity and function of certain repair pathways. In this review, we highlight the roles and consequences of G4 DNA structures for DNA repair initiation, processing, and the efficiency of various DNA repair pathways.

Keywords:  G-quadruplex; genome instability; homologous recombination; non-homologous end joining; nucleotide excision repair; translesion synthesis

DOI:  https://doi.org/10.3390/ijms222212599
Crit Rev Oncol Hematol. 2021 Nov 17. pii: S1040-8428(21)00326-7. [Epub ahead of print] 103539

Targeting the DNA damage response: PARP inhibitors and new perspectives in the landscape of cancer treatment.

Sofia Genta, Federica Martorana, Anastasios Stathis, Ilaria Colombo.

  Cancer derives from alterations of pathways responsible for cell survival, differentiation and proliferation. Dysfunctions of mechanisms protecting genome integrity can promote oncogenesis but can also be exploited as therapeutic target. Poly-ADP-Ribose-Polymerase (PARP)-inhibitors, the first approved targeted agents able to tackle DNA damage response (DDR), have demonstrated antitumor activity, particularly when homologous recombination impairment is present. Despite the relevant results achieved, a large proportion of patients fail to obtain durable responses. The development of innovative treatments, able to overcome resistance and ensure long-lasting benefit for a wider population is still an unmet need. Moreover, improvement in biomarker assays is necessary to properly identify patients who can benefit from DDR targeting agents. Here we summarize the main DDR pathways, explain the current role of PARP inhibitors in cancer therapy and illustrate new therapeutic strategies targeting the DDR, focusing on the combinations of PARP inhibitors with other agents and on cell-cycle checkpoint inhibitors.

Keywords:  DNA damage response; PARP inhibitors; PARP inhibitors resistance; cell-cycle checkpoint inhibitors; homologous recombination

DOI:  https://doi.org/10.1016/j.critrevonc.2021.103539
Int J Mol Sci. 2021 Nov 12. pii: 12254. [Epub ahead of print]22(22):

Genomic Instability and Cancer Risk Associated with Erroneous DNA Repair.

Ken-Ichi Yoshioka, Rika Kusumoto-Matsuo, Yusuke Matsuno, Masamichi Ishiai.

  Many cancers develop as a consequence of genomic instability, which induces genomic rearrangements and nucleotide mutations. Failure to correct DNA damage in DNA repair defective cells, such as in BRCA1 and BRCA2 mutated backgrounds, is directly associated with increased cancer risk. Genomic rearrangement is generally a consequence of erroneous repair of DNA double-strand breaks (DSBs), though paradoxically, many cancers develop in the absence of DNA repair defects. DNA repair systems are essential for cell survival, and in cancers deficient in one repair pathway, other pathways can become upregulated. In this review, we examine the current literature on genomic alterations in cancer cells and the association between these alterations and DNA repair pathway inactivation and upregulation.

Keywords:  chromosomal instability (CIN); genomic instability; homologous recombination (HR); microhomology-mediated end-joining (MMEJ); microsatellite instability (MSI); mismatch repair (MMR); non-homologous end-joining (NHEJ); nucleotide excision repair (NER)

DOI:  https://doi.org/10.3390/ijms222212254
Genes Dev. 2021 Nov 24.

Replication protein A associates with nucleolar R loops and regulates rRNA transcription and nucleolar morphology.

Shuang Feng, James L Manley.

  The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated "promoter and pre-rRNA antisense" RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.

Keywords:  R loop; RNA polymerase I (RNAP I); RNaseH1; replication protein A (RPA); senataxin (SETX)

DOI:  https://doi.org/10.1101/gad.348858.121
J Clin Invest. 2021 Nov 23. pii: e149711. [Epub ahead of print]

The cardiomyocyte disrupts pyrimidine biosynthesis in non-myocytes to regulate heart repair.

Shen Li, Tomohiro Yokota, Ping Wang, Johanna Ten Hoeve, Feiyang Ma, Thuc M Le, Evan R Abt, Yonggang Zhou, Rimao Wu, Maxine Nanthavongdouangsy, Abraham Rodriguez, Yijie Wang, Yen-Ju Lin, Hayato Muranaka, Mark Sharpley, Demetrios T Braddock, Vicky E MacRae, Utpal Banerjee, Pei-Yu Chiou, Marcus Seldin, Dian Huang, Michael Teitell, Ilya Gertsman, Michael Jung, Steven J Bensinger, Robert Damoiseaux, Kym Faull, Matteo Pellegrini, Aldons Lusis, Thomas G Graeber, Caius G Radu, Arjun Deb.

  Various population of cells are recruited to the heart after cardiac injury but little is known about whether the cardiomyocyte directly regulates heart repair. In a murine model of ischemic cardiac injury, we demonstrate that the cardiomyocyte plays a pivotal role in heart repair by regulating nucleotide metabolism and fates of non-myocytes. Cardiac injury induced the expression of the ectonucleotidase ENPP1 that hydrolyzes extracellular ATP to form AMP. In response to AMP, the cardiomyocyte released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at OMP synthesis step, induced genotoxic stress and a p53 mediated cell death of cycling non-myocytes. As non-myocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors on small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in non-myocyte cells, augmented cardiac repair and post infarct heart function. These observations demonstrate that the cardiac muscle cell by releasing adenine and specific nucleosides after heart injury regulates pyrimidine metabolism in non-muscle cells and provide insight into how inter-cellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Keywords:  Cardiology; Cardiovascular disease

DOI:  https://doi.org/10.1172/JCI149711
Biochim Biophys Acta Rev Cancer. 2021 Nov 17. pii: S0304-419X(21)00159-1. [Epub ahead of print] 188661

Genomic instability, inflammatory signaling and response to cancer immunotherapy.

Mengting Chen, Renske Linstra, Marcel A T M Van Vugt.

  Genomic and chromosomal instability are hallmarks of cancer and shape the genomic composition of cancer cells, thereby determining their behavior and response to treatment. Various genetic and epigenetic alterations in cancer have been linked to genomic instability, including DNA repair defects, oncogene-induced replication stress, and spindle assembly checkpoint malfunction. A consequence of genomic and chromosomal instability is the leakage of DNA from the nucleus into the cytoplasm, either directly or through the formation and subsequent rupture of micronuclei. Cytoplasmic DNA subsequently activates cytoplasmic DNA sensors, triggering downstream pathways, including a type I interferon response. This inflammatory signaling has pleiotropic effects, including enhanced anti-tumor immunity and potentially results in sensitization of cancer cells to immune checkpoint inhibitors. However, cancers frequently evolve mechanisms to avoid immune clearance, including suppression of inflammatory signaling. In this review, we summarize inflammatory signaling pathways induced by various sources of genomic instability, adaptation mechanisms that suppress inflammatory signaling, and implications for cancer immunotherapy.

Keywords:  Chromosomal instability; DNA damage repair; Homologous recombination; Immune checkpoint inhibition; Type I interferon; cGAS/STING pathway

DOI:  https://doi.org/10.1016/j.bbcan.2021.188661
Genes (Basel). 2021 Nov 05. pii: 1763. [Epub ahead of print]12(11):

Regulation of the Fanconi Anemia DNA Repair Pathway by Phosphorylation and Monoubiquitination.

Masamichi Ishiai.

  The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via phosphorylation and monoubiquitination.

Keywords:  ATM; ATR; DNA repair; Fanconi anemia; interstrand crosslink; phosphorylation; ubiquitination

DOI:  https://doi.org/10.3390/genes12111763
Nature. 2021 Nov 24.

Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response.

Lee Mulderrig, Juan I Garaycoechea, Zewen K Tuong, Christopher L Millington, Felix A Dingler, John R Ferdinand, Liam Gaul, John A Tadross, Mark J Arends, Stephen O'Rahilly, Gerry P Crossan, Menna R Clatworthy, Ketan J Patel.

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.

DOI: https://doi.org/10.1038/s41586-021-04133-7
Biomedicines. 2021 Oct 21. pii: 1512. [Epub ahead of print]9(11):

Role of PARP in TNBC: Mechanism of Inhibition, Clinical Applications, and Resistance.

Desh Deepak Singh, Amna Parveen, Dharmendra Kumar Yadav.

  Triple-negative breast cancer is a combative cancer type with a highly inflated histological grade that leads to poor theragnostic value. Gene, protein, and receptor-specific targets have shown effective clinical outcomes in patients with TNBC. Cells are frequently exposed to DNA-damaging agents. DNA damage is repaired by multiple pathways; accumulations of mutations occur due to damage to one or more pathways and lead to alterations in normal cellular mechanisms, which lead to development of tumors. Advances in target-specific cancer therapies have shown significant momentum; most treatment options cause off-target toxicity and side effects on healthy tissues. PARP (poly(ADP-ribose) polymerase) is a major protein and is involved in DNA repair pathways, base excision repair (BER) mechanisms, homologous recombination (HR), and nonhomologous end-joining (NEJ) deficiency-based repair mechanisms. DNA damage repair deficits cause an increased risk of tumor formation. Inhibitors of PARP favorably kill cancer cells in BRCA-mutations. For a few years, PARPi has shown promising activity as a chemotherapeutic agent in BRCA1- or BRCA2-associated breast cancers, and in combination with chemotherapy in triple-negative breast cancer. This review covers the current results of clinical trials testing and future directions for the field of PARP inhibitor development.

Keywords:  DNA damage repair; PARP (poly(ADP-ribose) polymerase); TNBC; breast cancer; signaling pathway; therapeutic target

DOI:  https://doi.org/10.3390/biomedicines9111512
Cells. 2021 Nov 20. pii: 3254. [Epub ahead of print]10(11):

Effects of 5',8'-Cyclo-2'-Deoxypurines on the Base Excision Repair of Clustered DNA Lesions in Nuclear Extracts of the XPC Cell Line.

Julia Kaźmierczak-Barańska, Karolina Boguszewska, Michał Szewczuk, Bolesław T Karwowski.

  Clustered DNA lesions (CDL) containing 5',8-cyclo-2'-deoxypurines (cdPus) are an example of extensive abnormalities occurring in the DNA helix and may impede cellular repair processes. The changes in the efficiency of nuclear base excision repair (BER) were investigated using (a) two cell lines, one of the normal skin fibroblasts as a reference (BJ) and the second from Xeroderma pigmentosum patients' skin (XPC), and (b) synthetic oligonucleotides with single- and double-stranded CDL (containing 5',8-cyclo-2'-deoxyadenosine (cdA) and the abasic (AP) site at various distances between lesions). The nuclear BER has been observed and the effect of both cdA isomers (5'R and 5'S) presence in the DNA was tested. CdPus affected the repair of the second lesion within the CDL. The BER system more efficiently processed damage in the vicinity of the ScdA isomer and changes located in the 3'-end direction for dsCDL and in the 5'-end direction for ssCDL. The presented study is the very first investigation of the repair processes of the CDL containing cdPu considering cells derived from a Xeroderma pigmentosum patient.

Keywords:  BER; BJ; DNA damage; DNA repair; XPC; cdA; clustered DNA lesions

DOI:  https://doi.org/10.3390/cells10113254