bims-numges 2021-05-23 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2021–05–23
28 papers selected by
Sean Rudd, Karolinska Institutet

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.
Selective Inhibition of DNA Polymerase β by a Covalent Inhibitor.
The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.
Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.
Replication initiation: Implications in genome integrity.
Non-canonical regulation of homologous recombination DNA repair by the USP9X deubiquitinase.
RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner.
Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.
APOBEC3G Rescues Cells from the Deleterious Effects of DNA Damage.
Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.
MIF is a 3' flap nuclease that facilitates DNA replication and promotes tumor growth.
Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours.
A functional in vitro cell-free system for studying DNA repair in isolated nuclei.
The role of α tubulin acetyltransferase αTAT1 in DNA damage response.
Ku70 suppresses alternative end joining in G1-arrested progenitor B cells.
Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions.
Timely upstream events regulating nucleotide excision repair by ubiquitin-proteasome system: ubiquitin guides the way.
Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae.
The role of homologous recombination deficiency testing in ovarian cancer and its clinical implications: do we need it?
CSB cooperates with SMARCAL1 to maintain telomere stability in ALT cells.
TDP-43 dysfunction results in R-loop accumulation and DNA replication defects.
Small-molecule inhibition of APE1 induces apoptosis, pyroptosis, and necroptosis in non-small cell lung cancer.
Synthesis, In-vitro evaluation and molecular docking studies of oxoindolin phenylhydrazine carboxamides as potent and selective inhibitors of ectonucleoside triphosphate diphosphohydrolase (NTPDase).
Chromatin-associated MRN complex protects highly transcribing genes from genomic instability.
The Mre11-Rad50-Nbs1 complex mediates the robust recruitment of Polo to DNA lesions during mitosis.
A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery.
Cytidine deaminase can deaminate fused pyrimidine ribonucleosides.

J Biol Chem. 2021 May 14. pii: S0021-9258(21)00573-1. [Epub ahead of print] 100780

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.

Kaito Mimura, Jun-Ichi Sakamaki, Hideaki Morishita, Masahito Kawazu, Hiroyuki Mano, Noboru Mizushima.

  Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

Keywords:  CRISPR/Cas; mammalian target of rapamycin (mTOR); nucleoside/nucleotide biosynthesis; nucleoside/nucleotide metabolism; nucleotide; phosphoribosylformylglycinamidine synthase (PFAS); tuberous sclerosis complex (TSC)

DOI:  https://doi.org/10.1016/j.jbc.2021.100780
J Am Chem Soc. 2021 May 20.

Selective Inhibition of DNA Polymerase β by a Covalent Inhibitor.

Shelby C Yuhas, Daniel J Laverty, Huijin Lee, Ananya Majumdar, Marc M Greenberg.

DNA polymerase β (Pol β) plays a vital role in DNA repair and has been closely linked to cancer. Selective inhibitors of this enzyme are lacking. Inspired by DNA lesions produced by antitumor agents that inactivate Pol β, we have undertaken the development of covalent small-molecule inhibitors of this enzyme. Using a two-stage process involving chemically synthesized libraries, we identified a potent irreversible inhibitor (14) of Pol β (KI = 1.8 ± 0.45 μM, kinact = (7.0 ± 1.0) × 10-3 s-1). Inhibitor 14 selectively inactivates Pol β over other DNA polymerases. LC-MS/MS analysis of trypsin digests of Pol β treated with 14 identified two lysines within the polymerase binding site that are covalently modified, one of which was previously determined to play a role in DNA binding. Fluorescence anisotropy experiments show that pretreatment of Pol β with 14 prevents DNA binding. Experiments using a pro-inhibitor (pro-14) in wild type mouse embryonic fibroblasts (MEFs) indicate that the inhibitor (5 μM) is itself not cytotoxic but works synergistically with the DNA alkylating agent, methylmethanesulfonate (MMS), to kill cells. Moreover, experiments in Pol β null MEFs indicate that pro-14 is selective for the target enzyme. Finally, pro-14 also works synergistically with MMS and bleomycin to kill HeLa cells. The results suggest that pro-14 is a potentially useful tool in studies of the role of Pol β in disease.

DOI: https://doi.org/10.1021/jacs.1c02453
Nucleic Acids Res. 2021 May 21. pii: gkab371. [Epub ahead of print]

The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Denis A Kiktev, Margaret Dominska, Tony Zhang, Joseph Dahl, Elena I Stepchenkova, Piotr Mieczkowski, Peter M Burgers, Scott Lujan, Adam Burkholder, Thomas A Kunkel, Thomas D Petes.

Iron-sulfur clusters (4Fe-4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol δ) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol δ are the observations that Pol δ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.

DOI: https://doi.org/10.1093/nar/gkab371
J Cell Sci. 2020 Jan 01. pii: jcs.241463. [Epub ahead of print]

Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.

Karolina Losenkova, Mariachiara Zuccarini, Marika Karikoski, Juha Laurila, Detlev Boison, Sirpa Jalkanen, Gennady G Yegutkin.

  Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects and becomes an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprised of (a) the ectoenzymatic breakdown of ATP via sequential nucleotide pyrophosphatase/phosphodiesterase-1, ecto-5'-nucleotidase/CD73 and adenosine deaminase reactions, and ATP re-synthesis through counteracting adenylate kinase and nucleoside diphosphokinase; (b) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (c) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 hours triggered ∼2-fold up-regulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.

Keywords:  Adenosine kinase; Adenosine metabolism; Breast tumor xenograft; CD39; Cancer cells; Ecto-5’-nucleotidase/CD73; Hypoxia

DOI:  https://doi.org/10.1242/jcs.241463
J Vis Exp. 2021 Apr 28.

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.

Julia O'Sullivan, Sofiane Y Mersaoui, Guy Poirier, Jean-Yves Masson.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2'-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

DOI: https://doi.org/10.3791/62553
DNA Repair (Amst). 2021 May 11. pii: S1568-7864(21)00087-2. [Epub ahead of print]103 103131

Replication initiation: Implications in genome integrity.

Yo-Chuen Lin, Supriya G Prasanth.

  In every cell cycle, billions of nucleotides need to be duplicated within hours, with extraordinary precision and accuracy. The molecular mechanism by which cells regulate the replication event is very complicated, and the entire process begins way before the onset of S phase. During the G1 phase of the cell cycle, cells prepare by assembling essential replication factors to establish the pre-replicative complex at origins, sites that dictate where replication would initiate during S phase. During S phase, the replication process is tightly coupled with the DNA repair system to ensure the fidelity of replication. Defects in replication and any error must be recognized by DNA damage response and checkpoint signaling pathways in order to halt the cell cycle before cells are allowed to divide. The coordination of these processes throughout the cell cycle is therefore critical to achieve genomic integrity and prevent diseases. In this review, we focus on the current understanding of how the replication initiation events are regulated to achieve genome stability.

Keywords:  DNA replication; Genome stability; Origin Firing; Origin Recognition Complex; Origin licensing; Pre-replication complex

DOI:  https://doi.org/10.1016/j.dnarep.2021.103131
J Cell Sci. 2020 Jan 01. pii: jcs.233437. [Epub ahead of print]

Non-canonical regulation of homologous recombination DNA repair by the USP9X deubiquitinase.

Rachel O'Dea, Corrado Santocanale.

  In order to prevent the deleterious effects of genotoxic agents, cells have developed complex surveillance mechanisms and DNA repair pathways that allow them to maintain genome integrity. The Ubiquitin Specific Protease 9X (USP9X) contributes to genome stability during DNA replication and chromosome segregation. Depletion of USP9X leads to DNA double strand breaks, some of which are triggered by replication fork collapse. Here we identify USP9X as a novel regulator of homologous recombination (HR) DNA repair in human cells. Using a cellular HR reporter assay, irradiation induced focus formation assays, and colony formation assays we show that USP9X is required for efficient HR. Mechanistically, we show USP9X is important to sustain the expression levels of key HR factors, namely BRCA1 and RAD51 through a non-canonical regulation of their mRNA abundance. Intriguingly, we find that USP9X contribution to BRCA1 and RAD51 expression is independent of its known catalytic activity. Thus this work identifies USP9X as a regulator of HR, demonstrates a novel mechanism by which USP9X can regulate protein levels and provides insights in to the regulation of BRCA1 and RAD51 mRNA.

Keywords:  BRCA1; DNA repair; Deubiquitination; RAD51; USP9X

DOI:  https://doi.org/10.1242/jcs.233437
J Cell Sci. 2020 Jan 01. pii: jcs.240036. [Epub ahead of print]

RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner.

Yuichiro Saito, Junya Kobayashi, Masato T Kanemaki, Kenshi Komatsu.

  RIF1 controls both the DNA replication timing and DNA double-strand break (DSB) repair pathways to maintain genome integrity. However, it remains unclear how RIF1 links these two processes when exposed to ionizing radiation (IR). Here, we show that homologous-recombination repair (HRR) inhibition by RIF1 occurs in a dose-dependent manner and is controlled via DNA replication. RIF1 inhibits both DNA end resection and RAD51 accumulation after exposure to high doses of IR. Contrastingly, HRR inhibition by RIF1 is antagonized by BRCA1 at a low-dose IR exposure. At high IR doses, RIF1 suppresses replication initiation by dephosphorylating MCM helicase. Notably, the dephosphorylation of MCM helicase inhibits both DNA end resection and HRR even without RIF1. Thus, our data show the importance of active DNA replication for HRR and suggest a common suppression mechanism for DNA replication and HRR at high IR doses, both of which are controlled by RIF1.

Keywords:  DNA repair; Homologous recombination; RIF1; Radiation biology

DOI:  https://doi.org/10.1242/jcs.240036
Genome Res. 2021 May 17.

Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair.

Kaitlynne A Bohm, Amelia J Hodges, Wioletta Czaja, Kathiresan Selvam, Michael J Smerdon, Peng Mao, John J Wyrick.

Nucleosomes are a significant barrier to the repair of UV damage because they impede damage recognition by nucleotide excision repair (NER). The RSC and SWI/SNF chromatin remodelers function in cells to promote DNA access by moving or evicting nucleosomes, and both have been linked to NER in yeast. Here, we report genome-wide repair maps of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast cells lacking RSC or SWI/SNF activity. Our data indicate that SWI/SNF is not generally required for NER but instead promotes repair of CPD lesions at specific yeast genes. In contrast, mutation or depletion of RSC subunits causes a general defect in NER across the yeast genome. Our data indicate that RSC is required for repair not only in nucleosomal DNA but also in neighboring linker DNA and nucleosome-free regions (NFRs). Although depletion of the RSC catalytic subunit also affects base excision repair (BER) of N-methylpurine (NMP) lesions, RSC activity is less important for BER in linker DNA and NFRs. Furthermore, our data indicate that RSC plays a direct role in transcription-coupled NER (TC-NER) of transcribed DNA. These findings help to define the specific genomic and chromatin contexts in which each chromatin remodeler functions in DNA repair, and indicate that RSC plays a unique function in facilitating repair by both NER subpathways.

DOI: https://doi.org/10.1101/gr.274373.120
FEBS J. 2021 May 17.

APOBEC3G Rescues Cells from the Deleterious Effects of DNA Damage.

Alexander Botvinnik, Pushkar Shivam, Yoav Smith, Gunjan Sharma, Udy Olshevsky, Ofra Moshel, Zakhariya Manevitch, Nuria Climent, Harold Oliva, Elena Britan-Rosich, Moshe Kotler.

  Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (hA3G), a member of the APOBEC family, was described as an anti-HIV-1 restriction factor, deaminating reverse transcripts of the HIV-1 genome. Several types of cancer cells that express high levels of A3G, such as diffuse large B-cell lymphoma cells and glioblastomas, show enhanced cell survival after ionizing radiation (IR) and chemotherapy treatments. Previously, we showed that hA3G promotes (DNA) double-strand breaks repair in cultured cells and it rescues transgenic mice from lethal IR dose. Here we show that A3G rescues cells from the detrimental effects of DNA damage induced by ultraviolet radiation (UV) and by combined bromodeoxyuridine and UV treatments. The combined treatments stimulate the synthesis of cellular proteins, which are exclusively associated with A3G expression. These proteins participate mainly in nucleotide excision repair and homologous recombination DNA repair pathways. Our results implicate A3G inhibition as a potential strategy for increasing tumor cells sensitivity to genotoxic treatments.

Keywords:  DNA repair; UV-irradiation; cytidine deaminases; radiation-resistance

DOI:  https://doi.org/10.1111/febs.16025
Mol Cell. 2021 May 12. pii: S1097-2765(21)00357-9. [Epub ahead of print]

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

Evgeniia Prokhorova, Thomas Agnew, Anne R Wondisford, Michael Tellier, Nicole Kaminski, Danique Beijer, James Holder, Josephine Groslambert, Marcin J Suskiewicz, Kang Zhu, Julia M Reber, Sarah C Krassnig, Luca Palazzo, Shona Murphy, Michael L Nielsen, Aswin Mangerich, Dragana Ahel, Jonathan Baets, Roderick J O'Sullivan, Ivan Ahel.

  ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

Keywords:  ADP-ribosylation; ARH3/ADPRHL2; BRCA; DNA damage; PARG; PARP inhibitor; cancer; chromatin; neurodegeneration; telomere

DOI:  https://doi.org/10.1016/j.molcel.2021.04.028
Nat Commun. 2021 05 19. 12(1): 2954

MIF is a 3' flap nuclease that facilitates DNA replication and promotes tumor growth.

Yijie Wang, Yan Chen, Chenliang Wang, Mingming Yang, Yanan Wang, Lei Bao, Jennifer E Wang, BongWoo Kim, Kara Y Chan, Weizhi Xu, Emanuela Capota, Janice Ortega, Deepak Nijhawan, Guo-Min Li, Weibo Luo, Yingfei Wang.

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3' flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3' nuclease, excises flaps at the immediate 3' end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.

DOI: https://doi.org/10.1038/s41467-021-23264-z
Oncogenesis. 2021 May 15. 10(5): 41

Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours.

Madushan Fernando, Pascal H G Duijf, Martina Proctor, Alexander J Stevenson, Anna Ehmann, Shivam Vora, Dubravka Skalamera, Mark Adams, Brian Gabrielli.

Defective DNA repair is being demonstrated to be a useful target in cancer treatment. Currently, defective repair is identified by specific gene mutations, however defective repair is a common feature of cancers without these mutations. DNA damage triggers cell cycle checkpoints that are responsible for co-ordinating cell cycle arrest and DNA repair. Defects in checkpoint signalling components such as ataxia telangiectasia mutated (ATM) occur in a low proportion of cancers and are responsible for reduced DNA repair and increased genomic instability. Here we have investigated the AURKA-PLK1 cell cycle checkpoint recovery pathway that is responsible for exit from the G2 phase cell cycle checkpoint arrest. We demonstrate that dysregulation of PP6 and AURKA maintained elevated PLK1 activation to promote premature exit from only ATM, and not ATR-dependent checkpoint arrest. Surprisingly, depletion of the B55α subunit of PP2A that negatively regulates PLK1 was capable of overcoming ATM and ATR checkpoint arrests. Dysregulation of the checkpoint recovery pathway reduced S/G2 phase DNA repair efficiency and increased genomic instability. We found a strong correlation between dysregulation of the PP6-AURKA-PLK1-B55α checkpoint recovery pathway with signatures of defective homologous recombination and increased chromosomal instability in several cancer types. This work has identified an unrealised source of G2 phase DNA repair defects and chromosomal instability that are likely to be sensitive to treatments targeting defective repair.

DOI: https://doi.org/10.1038/s41389-021-00329-8
J Cell Sci. 2020 Jan 01. pii: jcs.240010. [Epub ahead of print]

A functional in vitro cell-free system for studying DNA repair in isolated nuclei.

Isabella Guardamagna, Elisabetta Bassi, Monica Savio, Paola Perucca, Ornella Cazzalini, Ennio Prosperi, Lucia A Stivala.

  Assessing DNA repair is an important endpoint to study the DNA damage response for investigating the biochemical mechanisms of this process and the efficacy of chemotherapy, which often uses DNA damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, they show some limitations mainly due to the lack of chromatin organization. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the correspondent damage-activated cytosolic extracts, containing biotin-16-dUTP, nuclei are able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 or DDB1, stimulated UVC induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different type of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.

Keywords:  Cell-free system; DDB2; DNA repair; Nucleotide Excision Repair

DOI:  https://doi.org/10.1242/jcs.240010
J Cell Sci. 2020 Jan 01. pii: jcs.246702. [Epub ahead of print]

The role of α tubulin acetyltransferase αTAT1 in DNA damage response.

Na Mi Ryu, Jung Min Kim.

  Lysine 40 acetylation of α tubulin (Ac-α tubulin) catalyzed by acetyltransferase αTAT1, marks stabilized microtubules. Recently, there is growing evidence to suggest the crosstalk between DNA damage response (DDR) and microtubule organization, we therefore investigated whether αTAT1 is involved in DDR. Following treatment with DNA damaging agents, increased levels of Ac-α tubulin were detected. We also observed significant induction of Ac-α tubulin after depletion of DNA repair proteins, suggesting that αTAT1 is positively regulated in response to DNA damage. Intriguingly, αTAT1 depletion decreased DNA damage-induced RPA phosphorylation and foci formation. Moreover, DNA damage -induced cell cycle arrest was significantly delayed in αTAT1-depleted cells, indicating defective checkpoint activation. The checkpoint defects by αTAT1 deficiency were restored by expression of wild-type αTAT1, but not by D157N αTAT1 (catalytically inactive αTAT1), indicating that the role of αTAT1 in the DDR is dependent on enzymatic activity. Furthermore, αTAT1-depleted DR-GFP U2OS cells resulted in a significant decrease in the frequency of homologous recombination repair. Collectively, our results suggest that αTAT1 may play an essential role in DNA damage checkpoints and DNA repair through its acetyltransferase activity.

Keywords:  Checkpoint activation; DNA damage response; Homologous recombination repair; Microtubule stability; α tubulin acetyltransferase αTAT1; α tubulin lysine (K40) acetylation

DOI:  https://doi.org/10.1242/jcs.246702
Proc Natl Acad Sci U S A. 2021 May 25. pii: e2103630118. [Epub ahead of print]118(21):

Ku70 suppresses alternative end joining in G1-arrested progenitor B cells.

Zhuoyi Liang, Vipul Kumar, Marie Le Bouteiller, Jeffrey Zurita, Josefin Kenrick, Sherry G Lin, Jiangman Lou, Jianqiao Hu, Adam Yongxin Ye, Cristian Boboila, Frederick W Alt, Richard L Frock.

  Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.

Keywords:  Cas9; DSB repair; G1 phase; V(D)J recombination; end joining

DOI:  https://doi.org/10.1073/pnas.2103630118
J Cell Biol. 2021 Jul 05. pii: e202006149. [Epub ahead of print]220(7):

Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions.

Ramhari Kumbhar, Anthony Sanchez, Jullian Perren, Fade Gong, David Corujo, Frank Medina, Sravan K Devanathan, Blerta Xhemalce, Andreas Matouschek, Marcus Buschbeck, Bethany A Buck-Koehntop, Kyle M Miller.

The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)-binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi's) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.

DOI: https://doi.org/10.1083/jcb.202006149
DNA Repair (Amst). 2021 May 12. pii: S1568-7864(21)00084-7. [Epub ahead of print]103 103128

Timely upstream events regulating nucleotide excision repair by ubiquitin-proteasome system: ubiquitin guides the way.

Anil K Chauhan, Yingming Sun, Qianzheng Zhu, Altaf A Wani.

  The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of multiple DNA repair pathways, including nucleotide excision repair (NER), which eliminates a broad variety of helix-distorting DNA lesions that can otherwise cause deleterious mutations and genomic instability. In mammalian NER, DNA damage sensors, DDB and XPC acting in global genomic NER (GG-NER), and, CSB and RNAPII acting in transcription-coupled NER (TC-NER) sub-pathways, undergo an array of post-translational ubiquitination at the DNA lesion sites. Accumulating evidence indicates that ubiquitination orchestrates the productive assembly of NER preincision complex by driving well-timed compositional changes in DNA damage-assembled sensor complexes. Conversely, the deubiquitination is also intimately involved in regulating the damage sensing aftermath, via removal of degradative ubiquitin modification on XPC and CSB to prevent their proteolysis for the factor recycling. This review summaries the relevant research efforts and latest findings in our understanding of ubiquitin-mediated regulation of NER and active participation by new regulators of NER, e.g., Cullin-Ring ubiquitin ligases (CRLs), ubiquitin-specific proteases (USPs) and ubiquitin-dependent segregase, valosin-containing protein (VCP)/p97. We project hypothetical step-by-step models in which VCP/p97-mediated timely extraction of damage sensors is integral to overall productive NER. The USPs and proteasome subtly counteract in fine-tuning the vital stability and function of NER damage sensors.

Keywords:  Nucleotide excision repair; Ultraviolet radiation; deubiquitination; ubiquitination

DOI:  https://doi.org/10.1016/j.dnarep.2021.103128
G3 (Bethesda). 2021 May 01. pii: jkab124. [Epub ahead of print]

Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae.

Roland Chanet, Dorothée Baïlle, Marie-Pierre Golinelli-Cohen, Sylvie Riquier, Olivier Guittet, Michel Lepoivre, Meng-Er Huang, Laurence Vernis.

B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

DOI: https://doi.org/10.1093/g3journal/jkab124
ESMO Open. 2021 May 17. pii: S2059-7029(21)00103-4. [Epub ahead of print]6(3): 100144

The role of homologous recombination deficiency testing in ovarian cancer and its clinical implications: do we need it?

N Y L Ngoi, D S P Tan.

  The recognition of homologous recombination deficiency (HRD) as a frequent feature of high-grade serous ovarian cancer (HGSOC) has transformed treatment paradigms. Poly(ADP-ribose) polymerase inhibitors (PARPis), developed based on the rationale of synthetic lethality that predicates antitumor efficacy in tumors harboring underlying HRD, now represents an important class of therapy for HGSOC. Recent data have drawn attention to the assessment of homologous recombination DNA repair (HRR) as a prognostic and predictive biomarker in HGSOC, leading to increasing debate on the optimal means of defining and evaluating HRD, both genotypically and phenotypically. At present, clinical-grade assays such as myChoice CDx and FoundationOne CDx are approved companion diagnostics which can identify patients with HRD-positive HGSOC by diagnosing a 'genomic scar' reflecting underlying genomic instability. Yet despite the rapid maturation of this field, tumoral HRD status has been recognized to be dynamic over time and with treatment pressure. In practice, this means that restoration of HRR through mechanisms of platinum and PARPi resistance are not adequately represented by genomic scar assays, and contribute toward discordance with clinical PARPi response, or lack-thereof. It is thus critical that HRD testing is optimized to address the controversies of diverse HRD testing methodology, appropriate thresholds for HRD identification, and relevant timepoints for HRD testing, in order to realize the potential for PARPis to maximally benefit patients with HGSOC. Here, we discuss the premise of HRD testing in HGSOC, current methodologies for HRD identification and their performance in the clinic, highlight upcoming strategies, and discuss the challenges faced in moving this field forward.

Keywords:  biomarkers; homologous recombination deficiency; ovarian cancer; poly(ADP-ribose) polymerase inhibitors

DOI:  https://doi.org/10.1016/j.esmoop.2021.100144
J Cell Sci. 2020 Jan 01. pii: jcs.234914. [Epub ahead of print]

CSB cooperates with SMARCAL1 to maintain telomere stability in ALT cells.

Emily Feng, Nicole L Batenburg, John R Walker, Angus Ho, Taylor R H Mitchell, Jian Qin, Xu-Dong Zhu.

  Elevated replication stress is evident at telomeres of about 10-15% of cancer cells, which maintain their telomeres via a homologous recombination (HR)-based mechanism, referred to as alternative lengthening of telomeres (ALT). How ALT cells resolve replication stress to support their growth remains incompletely characterized. Here we report that CSB promotes recruitment of HR repair proteins (MRN, BRCA1, BLM, RPA32) and POLD3 to ALT telomeres, a process that requires CSB's ATPase activity and is controlled by ATM- and CDK2-dependent phosphorylation. Loss of CSB stimulates telomeric recruitment of MUS81 and SLX4, components of the structure-specific MUS81-EME1-SLX1-SLX4 (MUS-SLX) endonuclease complex, suggesting that CSB restricts MUS-SLX-mediated processing of stalled forks at ALT telomeres. Loss of CSB coupled with depletion of SMARCAL1, a chromatin remodeler implicated in catalyzing regression of stalled forks, synergistically promotes not only telomeric recruitment of MUS81 but also the formation of fragile telomeres, the latter of which is reported to arise from fork stalling. These results altogether suggest that CSB-mediated HR repair and SMARCAL1-mediated fork regression cooperate to prevent stalled forks from being processed into fragile telomeres in ALT cells.

Keywords:  Alternative lengthening of telomeres (ALT); Cockayne syndrome group B protein (CSB); Homologous recombinational repair; Replication stress; SMARCAL1; Telomere fragility

DOI:  https://doi.org/10.1242/jcs.234914
J Cell Sci. 2020 Jan 01. pii: jcs.244129. [Epub ahead of print]

TDP-43 dysfunction results in R-loop accumulation and DNA replication defects.

Matthew Wood, Annabel Quinet, Yea-Lih Lin, Albert A Davis, Philippe Pasero, Yuna M Ayala, Alessandro Vindigni.

  TDP-43 is an RNA binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress. TDP-43 nucleic-acid binding and self-assembly activities are important in inhibiting R-loop accumulation and preserving normal DNA replication. We also find that TDP-43 cytoplasmic aggregation impairs TDP-43 function in R-loop regulation. Furthermore, increased R-loop accumulation and DNA damage is observed in neurons upon loss of TDP-43. Together, our findings indicate that TDP-43 function and normal protein homeostasis are critical in maintaining genomic stability through a co-transcriptional process that prevents aberrant R-loop accumulation. We propose that the increased R-loop formation and genomic instability associated with TDP-43 loss are linked to the pathogenesis of TDP-43 proteinopathies.

Keywords:  DNA Replication; R-loops; RNA:DNA hybrids; TDP-43; Tardbp

DOI:  https://doi.org/10.1242/jcs.244129
Cell Death Dis. 2021 May 18. 12(6): 503

Small-molecule inhibition of APE1 induces apoptosis, pyroptosis, and necroptosis in non-small cell lung cancer.

Kaili Long, Lili Gu, Lulu Li, Ziyu Zhang, Enjie Li, Yilan Zhang, Lingfeng He, Feiyan Pan, Zhigang Guo, Zhigang Hu.

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, which is responsible for the excision of apurinic sites (AP sites). In non-small cell lung cancer (NSCLC), APE1 is highly expressed and associated with poor patient prognosis. The suppression of APE1 could lead to the accumulation of unrepaired DNA damage in cells. Therefore, APE1 is viewed as an important marker of malignant tumors and could serve as a potent target for the development of antitumor drugs. In this study, we performed a high-throughput virtual screening of a small-molecule library using the three-dimensional structure of APE1 protein. Using the AP site cleavage assay and a cell survival assay, we identified a small molecular compound, NO.0449-0145, to act as an APE1 inhibitor. Treatment with NO.0449-0145 induced DNA damage, apoptosis, pyroptosis, and necroptosis in the NSCLC cell lines A549 and NCI-H460. This inhibitor was also able to impede cancer progression in an NCI-H460 mouse model. Moreover, NO.0449-0145 overcame both cisplatin- and erlotinib-resistance in NSCLC cell lines. These findings underscore the importance of APE1 as a therapeutic target in NSCLC and offer a paradigm for the development of small-molecule drugs that target key DNA repair proteins for the treatment of NSCLC and other cancers.

DOI: https://doi.org/10.1038/s41419-021-03804-7
Bioorg Chem. 2021 Apr 30. pii: S0045-2068(21)00334-5. [Epub ahead of print]112 104957

Synthesis, In-vitro evaluation and molecular docking studies of oxoindolin phenylhydrazine carboxamides as potent and selective inhibitors of ectonucleoside triphosphate diphosphohydrolase (NTPDase).

Saira Afzal, Mariya Al-Rashida, Abdul Hameed, Julie Pelletier, Jean Sévigny, Jamshed Iqbal.

  Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the hydrolysis of nucleoside triphosphate (NTP) and nucleoside diphosphates (NDP) to nucleoside monophosphate (NMP). Although, NTPDase inhibitors can act as potential drug candidates for the treatment of various diseases, there is lack of potent as well as selective inhibitors of NTPDases. The current study describes the synthesis of a number of carboxamide derivatives that were tested on recombinant human (h) NTPDases. The most promising inhibitors were 2h (h-NTPDase1, IC50: 0.12 ± 0.03 µM), 2d (h-NTPDase2, IC50: 0.15 ± 0.01 µM) and 2a (h-NTPDase3, IC50: 0.30 ± 0.04 µM; h-NTPDase8, IC50: 0.16 ± 0.02 µM). Four compounds (2e, 2f, 2g and 2h) were associated with the selective inhibition of h-NTPDase1 while 2b was identified as a selective h-NTPDase3 inhibitor. Considering the importance of NTPDase3 in the regulation of insulin release, the NTPDase3 inhibitors were further investigated to elucidate their role in the insulin release. The obtained data suggested that compound 2a was actively participating in regulating the insulin release without producing any effect on NTPDase3 mRNA. Moreover, the most potent inhibitors were docked within the active site of respective enzyme and the observed interactions were in compliance with in vitro results. Hence, these compounds can be used as pharmacological tool to further investigate the role of NTPDase3 coupled to insulin release.

Keywords:  Carboxamide; Isatin; Molecular docking; Nucleoside triphosphate diphosphohydrolase inhibitors; Oxoindolin

DOI:  https://doi.org/10.1016/j.bioorg.2021.104957
Sci Adv. 2021 May;pii: eabb2947. [Epub ahead of print]7(21):

Chromatin-associated MRN complex protects highly transcribing genes from genomic instability.

Kader Salifou, Callum Burnard, Poornima Basavarajaiah, Giuseppa Grasso, Marion Helsmoortel, Victor Mac, David Depierre, Céline Franckhauser, Emmanuelle Beyne, Xavier Contreras, Jérôme Dejardin, Sylvie Rouquier, Olivier Cuvier, Rosemary Kiernan.

MRN-MDC1 plays a central role in the DNA damage response (DDR) and repair. Using proteomics of isolated chromatin fragments, we identified DDR factors, such as MDC1, among those highly associating with a genomic locus upon transcriptional activation. Purification of MDC1 in the absence of exogenous DNA damage revealed interactions with factors involved in gene expression and RNA processing, in addition to DDR factors. ChIP-seq showed that MRN subunits, MRE11 and NBS1, colocalized throughout the genome, notably at TSSs and bodies of actively transcribing genes, which was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at MRE11/NBS1-bound genes. Prolonged MRE11 or NBS1 depletion induced single-nucleotide polymorphisms across actively transcribing MRN target genes. These data suggest that association of MRN with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.

DOI: https://doi.org/10.1126/sciadv.abb2947
J Cell Sci. 2020 Jan 01. pii: jcs.244442. [Epub ahead of print]

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

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

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

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

DOI:  https://doi.org/10.1242/jcs.244442
Sci Adv. 2021 May;pii: eabf0197. [Epub ahead of print]7(21):

A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery.

Kenichi Iwai, Tadahiro Nambu, Yukie Kashima, Jie Yu, Kurt Eng, Kazumasa Miyamoto, Kazuyo Kakoi, Masamitsu Gotou, Toshiyuki Takeuchi, Akifumi Kogame, Jessica Sappal, Saomi Murai, Hiroshi Haeno, Shun-Ichiro Kageyama, Osamu Kurasawa, Huifeng Niu, Karuppiah Kannan, Akihiro Ohashi.

Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.

DOI: https://doi.org/10.1126/sciadv.abf0197
Org Biomol Chem. 2021 May 21.

Cytidine deaminase can deaminate fused pyrimidine ribonucleosides.

Paul T Ludford, Yao Li, Shenghua Yang, Yitzhak Tor.

The tolerance of cytidine deaminase (CDA) to expanded heterocycles is explored via three fluorescent cytidine analogues, where the pyrimidine core is fused to three distinct five-membered heterocycles at the 5/6 positions. The reaction between CDA and each analogue is followed by absorption and emission spectroscopy, revealing shorter reaction times for all analogues than the native substrate. Pseudo-first order and Michaelis-Menten kinetic analyses provide insight into the enzymatic deamination reactions and assist in drawing comparison to established structure activity relationships. Finally, inhibitor screening modalities are created for each analogue and validated with zebularine and tetrahydrouridine, two known CDA inhibitors.

DOI: https://doi.org/10.1039/d1ob00705j