bims-numges 2022-01-30 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022–01–30
thirty-two papers selected by
Sean Rudd, Karolinska Institutet

Nucleotide biosynthesis links glutathione metabolism to ferroptosis sensitivity.
Aptamers Entirely Built from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy.
Proline-specific aminopeptidase P prevents replication-associated genome instability.
Beyond the Lesion: Back to High Fidelity DNA Synthesis.
Single Molecular Resolution to Monitor DNA Replication Fork Dynamics upon Stress by DNA Fiber Assay.
The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination.
ATR inhibitor AZD6738 (ceralasertib) exerts antitumor activity as a monotherapy and in combination with chemotherapy and the PARP inhibitor olaparib.
Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging.
Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection.
SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation.
The consequences of differential origin licensing dynamics in distinct chromatin environments.
Targeting PARP proteins in acute leukemia: DNA damage response inhibition and therapeutic strategies.
A Proximity Ligation Method to Detect Proteins Bound to Single-Stranded DNA after DNA End Resection at DNA Double-Strand Breaks.
When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer.
Miscoding and DNA Polymerase Stalling by Methoxyamine-Adducted Abasic Sites.
Top1p targeting by Fob1p at the ribosomal Replication Fork Barrier does not account for camptothecin sensitivity in Saccharomyces cerevisiae cells.
Dual-functional significance of ATM-mediated phosphorylation of spindle assembly checkpoint component Bub3 in mitosis and the DNA damage response.
Compartmentalization of telomeres through DNA-scaffolded phase separation.
Pharmacological Targeting of STING-Dependent IL-6 Production in Cancer Cells.
Mechanism of action of nucleotide excision repair machinery.
Structure of the human ATM kinase and mechanism of Nbs1 binding.
USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma.
A Novel Role for DNA-PK in Metabolism by Regulating Glycolysis in Castration Resistant Prostate Cancer.
Dose-dependent bioavailability and tissue distribution of the ATR inhibitor AZD6738 (ceralasertib) in mice.
Mapping molecular subtype specific alterations in breast cancer brain metastases identifies clinically relevant vulnerabilities.
Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal.
Olaparib Induced Senescence is Bypassed through G2/M Checkpoint Override in Olaparib Resistant Prostate Cancer.
Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase.
Homology-directed repair involves multiple strand invasion cycles in fission yeast.
Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools.
Targeting cellular iron homeostasis with ironomycin in diffuse large B cell lymphoma.
Elimination of Aicardi Goutières Syndrome Protein SAMHD1 Activates Cellular Innate Immunity and Suppresses SARS-CoV-2 Replication.

Life Sci Alliance. 2022 Apr;pii: e202101157. [Epub ahead of print]5(4):

Nucleotide biosynthesis links glutathione metabolism to ferroptosis sensitivity.

Amy Tarangelo, Jason Rodencal, Joon Tae Kim, Leslie Magtanong, Jonathan Z Long, Scott J Dixon.

Nucleotide synthesis is a metabolically demanding process essential for DNA replication and other processes in the cell. Several anticancer drugs that inhibit nucleotide metabolism induce apoptosis. How inhibition of nucleotide metabolism impacts non-apoptotic cell death is less clear. Here, we report that inhibition of nucleotide metabolism by the p53 pathway is sufficient to suppress the non-apoptotic cell death process of ferroptosis. Mechanistically, stabilization of wild-type p53 and induction of the p53 target gene CDKN1A (p21) leads to decreased expression of the ribonucleotide reductase (RNR) subunits RRM1 and RRM2 RNR is the rate-limiting enzyme of de novo nucleotide synthesis that reduces ribonucleotides to deoxyribonucleotides in a glutathione-dependent manner. Direct inhibition of RNR results in conservation of intracellular glutathione, limiting the accumulation of toxic lipid peroxides and preventing the onset of ferroptosis in response to cystine deprivation. These results support a mechanism linking p53-dependent regulation of nucleotide metabolism to non-apoptotic cell death.

DOI: https://doi.org/10.26508/lsa.202101157
J Am Chem Soc. 2022 Jan 24.

Aptamers Entirely Built from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy.

Lijuan Zhu, Jiapei Yang, Yuan Ma, Xinyuan Zhu, Chuan Zhang.

Owing to the specific and high binding affinity of aptamers to their targets, aptamer-drug conjugates (ApDCs) have emerged as a promising drug delivery system for targeted cancer therapy. However, in a conventional ApDC, the aptamer segment usually just serves as a targeting moiety, and only a limited number of drug molecules are sequentially conjugated to the oligonucleotide, giving a relatively low drug loading capacity. To address this challenge, herein we employ four clinically approved nucleoside analogues, including clofarabine (Clo), ara-guanosine (AraG), gemcitabine (Ge), and floxuridine (FdU), to replace all natural nucleosides in aptamer sequences, generating a series of whole drug-constituted DNA-like oligomers that are termed drugtamers. Similar to their parent aptamers, the obtained drugtamers maintain the targeting capability and can specifically bind to the target receptors overexpressed on the cancer cell surface. With 100% drug loading ratio, active targeting capability, and enzyme-mediated release of active therapeutics, our drugtamers can strongly induce the apoptosis of cancer cells and inhibit the tumor progression, which enables a new potential for a better targeted cancer therapy.

DOI: https://doi.org/10.1021/jacs.1c09574
PLoS Genet. 2022 Jan 26. 18(1): e1010025

Proline-specific aminopeptidase P prevents replication-associated genome instability.

Nicola Silva, Maikel Castellano-Pozo, Kenichiro Matsuzaki, Consuelo Barroso, Monica Roman-Trufero, Hannah Craig, Darren R Brooks, R Elwyn Isaac, Simon J Boulton, Enrique Martinez-Perez.

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1's role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.

DOI: https://doi.org/10.1371/journal.pgen.1010025
Front Mol Biosci. 2021 ;8 811540

Beyond the Lesion: Back to High Fidelity DNA Synthesis.

Joseph D Kaszubowski, Michael A Trakselis.

  High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases. Once stalled, the replisome becomes compromised and vulnerable to further DNA damage. One mechanism to relieve stalling is to recruit a translesion synthesis (TLS) Pol to rapidly synthesize over and past the damage. These TLS Pols have good specificities for the lesion but are less accurate when synthesizing opposite undamaged DNA, and so, mechanisms are needed to limit TLS Pol synthesis and recruit back a HiFi Pol to reestablish the replisome. The overall TLS process can be complicated with several cellular Pols, multifaceted protein contacts, and variable nucleotide incorporation kinetics all contributing to several discrete substitution (or template hand-off) steps. In this review, we highlight the mechanistic differences between distributive equilibrium exchange events and concerted contact-dependent switching by DNA Pols for insertion, extension, and resumption of high-fidelity synthesis beyond the lesion.

Keywords:  DNA damage; DNA lesion; DNA replication; PCNA; polymerase; substitution; switching; translesion synthesis

DOI:  https://doi.org/10.3389/fmolb.2021.811540
Bio Protoc. 2021 Dec 20. 11(24): e4269

Single Molecular Resolution to Monitor DNA Replication Fork Dynamics upon Stress by DNA Fiber Assay.

Wenpeng Liu.

  DNA replication always encounters numerous endogenous and exogenous stresses during the cell cycle. Measuring the cell responses to stress has primarily relied on cell survival and incorporation of radioactive dNTPs, which is limited in resolution. A higher resolution is required to monitor how replication and repair respond to these stresses. This protocol describes a procedure to monitor the length of new synthesized DNA in a single molecular resolution called DNA fiber assay. The fiber assay relies on labeling of nascent DNA with the nucleoside analog 5-Chloro-2'-deoxyuridine (CldU) and 5-Iodo-2'-deoxyuridine (IdU). We can visualize the labeled nascent DNA in single molecular resolution by immunostaining. By measuring labeled DNA length, the assay permits interrogation of replication speed at given conditions, end processing at the reversed fork, and fork restart after repair.

Keywords:  DNA fiber assay; Fork degradation; Fork restart; Fork speed; Nascent DNA strand degradation

DOI:  https://doi.org/10.21769/BioProtoc.4269
Front Genet. 2021 ;12 823943

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination.

Alice Libri, Timea Marton, Ludovic Deriano.

  DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

Keywords:  DNA double-strand break; DNA double-strand break repair pathway choice; DNA end resection; V(D)J recombination; homology-directed repair; non-homologous end-joining

DOI:  https://doi.org/10.3389/fgene.2021.823943
Cancer Res. 2022 Jan 25. pii: canres.2997.2021. [Epub ahead of print]

ATR inhibitor AZD6738 (ceralasertib) exerts antitumor activity as a monotherapy and in combination with chemotherapy and the PARP inhibitor olaparib.

Zena Wilson, Rajesh Odedra, Yann Wallez, Paul W G Wijnhoven, Adina M Hughes, Joe Gerrard, Gemma N Jones, Hannah Bargh-Dawson, Elane Brown, Lucy A Young, Mark J O'Connor, Alan Lau.

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2/M-cell cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication (BIR) and homologous recombination repair (HRR). In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM-pathway or that harbor putative drivers of replication stress such as CCNE1-amplification. This translated to in vivo anti-tumor activity, with tumor control requiring continuous dosing and free plasma exposures which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimisation of dose and schedules in vivo and showed superior anti-tumor activity at lower doses compared to that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice-daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3-5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice-daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2997
Biochem Biophys Res Commun. 2022 Jan 15. pii: S0006-291X(22)00055-9. [Epub ahead of print]594 57-62

Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging.

Vandana Singh, Pegah Johansson, Elina Ekedahl, Yii-Lih Lin, Ola Hammarsten, Fredrik Westerlund.

  DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

Keywords:  Base excision repair; Chemotherapy; DNA damage; Nick translation; Single molecule imaging; Single-strand breaks

DOI:  https://doi.org/10.1016/j.bbrc.2022.01.041
Life Sci Alliance. 2022 May;pii: e202101244. [Epub ahead of print]5(5):

Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection.

Martin Scherzer, Fosco Giordano, Maria Solé Ferran, Lena Ström.

Homologous recombination enables cells to overcome the threat of DNA double-strand breaks (DSBs), allowing for repair without the loss of genetic information. Central to the homologous recombination repair process is the de novo loading of cohesin around a DSB by its loader complex Scc2/4. Although cohesin's DSB accumulation has been explored in numerous studies, the prerequisites for Scc2/4 recruitment during the repair process are still elusive. To address this question, we combine chromatin immunoprecipitation-qPCR with a site-specific DSB in vivo, in Saccharomyces cerevisiae We find that Scc2 DSB recruitment relies on γH2A and Tel1, but as opposed to cohesin, not on Mec1. We further show that the binding of Scc2, which emanates from the break site, depends on and coincides with DNA end resection. Absence of chromatin remodeling at the DSB affects Scc2 binding and DNA end resection to a comparable degree, further indicating the latter to be a major driver for Scc2 recruitment. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and subsequent loading of cohesin.

DOI: https://doi.org/10.26508/lsa.202101244
FEBS Open Bio. 2022 Jan 24.

SV40 T antigen helicase domain regions responsible for oligomerisation regulate Okazaki fragment synthesis initiation.

Nichodemus O Onwubiko, Felicia Scheffel, Ingrid Tessmer, Heinz Peter Nasheuer.

  The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that whilst Tag131-627 (V350E/P417D) and Tag131-627 (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag1-708 and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules, but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regards to Okazaki fragment initiation.

Keywords:  DNA polymerase α-primase (Pol α); Eukaryotic DNA replication; Initiation reaction; Okazaki fragment synthesis; Replication protein A (RPA); SV40 large T antigen

DOI:  https://doi.org/10.1002/2211-5463.13373
Nucleic Acids Res. 2022 Jan 26. pii: gkac003. [Epub ahead of print]

The consequences of differential origin licensing dynamics in distinct chromatin environments.

Liu Mei, Katarzyna M Kedziora, Eun-Ah Song, Jeremy E Purvis, Jeanette Gowen Cook.

Eukaryotic chromosomes contain regions of varying accessibility, yet DNA replication factors must access all regions. The first replication step is loading MCM complexes to license replication origins during the G1 cell cycle phase. It is not yet known how mammalian MCM complexes are adequately distributed to both accessible euchromatin regions and less accessible heterochromatin regions. To address this question, we combined time-lapse live-cell imaging with immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in euchromatin and heterochromatin throughout G1. We report here that MCM loading in euchromatin is faster than that in heterochromatin in early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. This differential acceleration allows both chromatin types to begin S phase with similar concentrations of loaded MCM. The different loading dynamics require ORCA-dependent differences in origin recognition complex distribution. A consequence of heterochromatin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus, G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

DOI: https://doi.org/10.1093/nar/gkac003
J Hematol Oncol. 2022 Jan 22. 15(1): 10

Targeting PARP proteins in acute leukemia: DNA damage response inhibition and therapeutic strategies.

Antonella Padella, Andrea Ghelli Luserna Di Rorà, Giovanni Marconi, Martina Ghetti, Giovanni Martinelli, Giorgia Simonetti.

  The members of the Poly(ADP-ribose) polymerase (PARP) superfamily are involved in several biological processes and, in particular, in the DNA damage response (DDR). The most studied members, PARP1, PARP2 and PARP3, act as sensors of DNA damages, in order to activate different intracellular repair pathways, including single-strand repair, homologous recombination, conventional and alternative non-homologous end joining. This review recapitulates the functional role of PARPs in the DDR pathways, also in relationship with the cell cycle phases, which drives our knowledge of the mechanisms of action of PARP inhibitors (PARPi), encompassing inhibition of single-strand breaks and base excision repair, PARP trapping and sensitization to antileukemia immune responses. Several studies have demonstrated a preclinical activity of the current available PARPi, olaparib, rucaparib, niraparib, veliparib and talazoparib, as single agent and/or in combination with cytotoxic, hypomethylating or targeted drugs in acute leukemia, thus encouraging the development of clinical trials. We here summarize the most recent preclinical and clinical findings and discuss the synthetic lethal interactions of PARPi in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite the low frequency of genomic alterations of PARP and other DDR-related genes in acute leukemia, selective vulnerabilities have been reported in several disease subgroups, along with a "BRCAness phenotype." AML carrying the RUNX1-RUNX1T1 or PML-RARA fusion genes or mutations in signaling genes (FLT3-ITD in combination with TET2 or TET2 and DNMT3A deficiency), cohesin complex members (STAG2), TP53 and BCOR as co-occurring lesions, IDH1/2 and ALL cases expressing the TCF3-HLF chimera or TET1 was highly sensitive to PARPi in preclinical studies. These data, along with the warning coming from the observation of cases of therapy-related myeloid malignancies among patients receiving PARPi for solid tumors treatment, indicate that PARPi represents a promising strategy in a personalized medicine setting. The characterization of the clonal and subclonal genetic background and of the DDR functionality is crucial to select acute leukemia patients that will likely benefit of PARPi-based therapeutic regimens.

Keywords:  Acute lymphoblastic leukemia; Acute myeloid leukemia; Biomarkers; Clinical trials; DNA damage response; PARP; Preclinical studies; Synthetic lethality

DOI:  https://doi.org/10.1186/s13045-022-01228-0
Methods Protoc. 2021 Dec 29. pii: 3. [Epub ahead of print]5(1):

A Proximity Ligation Method to Detect Proteins Bound to Single-Stranded DNA after DNA End Resection at DNA Double-Strand Breaks.

Faith C Fowler, Jessica K Tyler.

  After a DNA double-strand break, cells utilize either non-homologous end joining or homologous recombination to repair the broken DNA ends. Homologous recombination requires extensive nucleolytic processing of one of the DNA strands, resulting in long stretches of 3' single-strand DNA overhangs. Typically, single-stranded DNA is measured using immunofluorescence microscopy to image the foci of replication protein A, a single-stranded DNA-binding protein. Microscopy analysis of bromodeoxyuridine foci under nondenaturing conditions has also been used to measure single-stranded DNA. Here, we describe a proximity ligation assay which uses genome-wide bromodeoxyuridine incorporation to label single-stranded DNA in order to measure the association of a protein of interest with single-stranded DNA. This method is advantageous over traditional foci analysis because it is more direct and specific than traditional foci co-localization microscopy methods, uses only one color channel, and can reveal protein-single-stranded DNA interactions that are rare and potentially undetectable using traditional microscopy methods. We show here the association of replication protein A and bromodeoxyuridine as proof-of-concept.

Keywords:  DNA end resection; double-strand break repair; proximity ligation assay; single-stranded DNA

DOI:  https://doi.org/10.3390/mps5010003
Front Mol Biosci. 2021 ;8 815845

When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer.

Denisse Carvajal-Maldonado, Lea Drogalis Beckham, Richard D Wood, Sylvie Doublié.

  DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3'-5' exonuclease, 5' nuclease, or end-trimming nuclease) or lyase (5' dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.

Keywords:  DNA polymerases; DNA repair; lyase activity; nuclease activity; nucleotidyl transfer; proofreading

DOI:  https://doi.org/10.3389/fmolb.2021.815845
Chem Res Toxicol. 2022 Jan 28.

Miscoding and DNA Polymerase Stalling by Methoxyamine-Adducted Abasic Sites.

Anna V Yudkina, Dmitry O Zharkov.

Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the "A-rule", preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of Escherichia coli DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases δ and ε, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol κ.

DOI: https://doi.org/10.1021/acs.chemrestox.1c00359
MicroPubl Biol. 2022 ;2022

Top1p targeting by Fob1p at the ribosomal Replication Fork Barrier does not account for camptothecin sensitivity in Saccharomyces cerevisiae cells.

Pardis Pourali, Philippe Pasero, Benjamin Pardo.

Camptothecin (CPT) is a specific inhibitor of the DNA topoisomerase I (Top1p), currently used in cancer therapy, which induces DNA damage and cell death. Top1p is highly active at the repeated ribosomal DNA locus (rDNA) to relax DNA supercoiling caused by elevated transcription and replication occurring in opposite directions. Fob1p interacts with, and stabilizes, Top1p at the rDNA Replication Fork Barrier (rRFB), where replication and transcription converge. Here, we have investigated if the absence of Fob1p and the consequent loss of Top1p specific targeting to the rRFB impact the sensitivity and the cell cycle progression of wild-type cells to CPT. We have also investigated the consequences of the absence of Fob1p in rad52∆ mutants, which are affected in the repair of CPT-induced DNA damage by homologous recombination. The results show that CPT sensitivity and the global cell cycle progression in cells exposed to CPT is not changed in the absence of Fob1p. Moreover, we have observed in fob1∆ cells treated with CPT that the homologous recombination factor Rad52p still congregates in the shape of foci in the nucleolus, which hosts the rDNA. This suggests that, in the absence of Fob1p, Top1p is still recruited to the rDNA, presumably at sequences other than the rRFB, and its inhibition by CPT leads to recombination events.

DOI: https://doi.org/10.17912/micropub.biology.000514
J Biol Chem. 2022 Jan 24. pii: S0021-9258(22)00072-2. [Epub ahead of print] 101632

Dual-functional significance of ATM-mediated phosphorylation of spindle assembly checkpoint component Bub3 in mitosis and the DNA damage response.

Mingming Xiao, Siyue Zhang, Zhuang Liu, Yaqi Mo, Han Wang, Xu Zhao, Xue Yang, Rebecca J Boohaker, Yang Chen, Yamei Han, Hong Liu, Bo Xu.

  Both the DNA damage response (DDR) and the mitotic checkpoint are critical for the maintenance of genomic stability. Among proteins involved in these processes, the Ataxia-Telangiectasia Mutated (ATM) kinase is required for both activation of the DDR and the spindle assembly checkpoint (SAC). In mitosis without DNA damage, the enzymatic activity of ATM is enhanced; however, substrates of ATM in mitosis are unknown. Using Stable Isotope Labeled Amino Acid in cell culture (SILAC)-mass spectrometry analysis, we identified a number of proteins that can potentially be phosphorylated by ATM during mitosis. This list is highly enriched in proteins involved in cell cycle regulation and the DDR. Among them, we further validated that ATM phosphorylated Budding Uninhibited by Benzimidazoles 3 (Bub3), a major component of the SAC, on serine 135 both in vitro and in vivo. During mitosis, this phosphorylation promoted activation of another SAC component, Bub1. Mutation of Bub3 serine 135 to alanine led to a defect in SAC activation. Furthermore, we found that ATM-mediated phosphorylation of Bub3 on serine 135 was also induced by ionizing radiation-induced DNA damage. However, this event resulted in independent signaling involving interaction with the Ku70-Ku80-DNA-PKcs sensor/kinase complex, leading to efficient non-homologous end joining repair. Taken together, we highlight the functional significance of the crosstalk between the kinetochore-oriented signal and double strand break repair pathways via ATM phosphorylation of Bub3 on serine 135.

Keywords:  ATM; Bub3; DNA damage response; Mitosis; phosphorylation

DOI:  https://doi.org/10.1016/j.jbc.2022.101632
Dev Cell. 2022 Jan 24. pii: S1534-5807(21)01037-6. [Epub ahead of print]57(2): 277-290.e9

Compartmentalization of telomeres through DNA-scaffolded phase separation.

Amanda Jack, Yoonji Kim, Amy R Strom, Daniel S W Lee, Byron Williams, Jeffrey M Schaub, Elizabeth H Kellogg, Ilya J Finkelstein, Luke S Ferro, Ahmet Yildiz, Clifford P Brangwynne.

  Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

Keywords:  DNA repair; chromatin organization; phase separation; shelterin; telomeres

DOI:  https://doi.org/10.1016/j.devcel.2021.12.017
Front Cell Dev Biol. 2021 ;9 709618

Pharmacological Targeting of STING-Dependent IL-6 Production in Cancer Cells.

Sumaiah S Al-Asmari, Aleksandra Rajapakse, Tomalika R Ullah, Geneviève Pépin, Laura V Croft, Michael P Gantier.

  Activation of the STING pathway upon genotoxic treatment of cancer cells has been shown to lead to anti-tumoral effects, mediated through the acute production of interferon (IFN)-β. Conversely, the pathway also correlates with the expression of NF-κB-driven pro-tumorigenic genes, but these associations are only poorly defined in the context of genotoxic treatment, and are thought to correlate with a chronic engagement of the pathway. We demonstrate here that half of the STING-expressing cancer cells from the NCI60 panel rapidly increased expression of pro-tumorigenic IL-6 upon genotoxic DNA damage, often independent of type-I IFN responses. While preferentially dependent on canonical STING, we demonstrate that genotoxic DNA damage induced by camptothecin (CPT) also drove IL-6 production through non-canonical STING signaling in selected cancer cells. Consequently, pharmacological inhibition of canonical STING failed to broadly inhibit IL-6 production induced by CPT, although this could be achieved through downstream ERK1/2 inhibition. Finally, prolonged inhibition of canonical STING signaling was associated with increased colony formation of MG-63 cells, highlighting the duality of STING signaling in also restraining the growth of selected cancer cells. Collectively, our findings demonstrate that genotoxic-induced DNA damage frequently leads to the rapid production of pro-tumorigenic IL-6 in cancer cells, independent of an IFN signature, through canonical and non-canonical STING activation; this underlines the complexity of STING engagement in human cancer cells, with frequent acute pro-tumorigenic activities induced by DNA damage. We propose that inhibition of ERK1/2 may help curb such pro-tumorigenic responses to DNA-damage, while preserving the anti-proliferative effects of the STING-interferon axis.

Keywords:  DNA damage; ERK1/2; IL-6; Non-canonical STING; STING; STING inhibitor; cancer

DOI:  https://doi.org/10.3389/fcell.2021.709618
Biochem Soc Trans. 2022 Jan 25. pii: BST20210246. [Epub ahead of print]

Mechanism of action of nucleotide excision repair machinery.

Areetha D'Souza, Alexandra M Blee, Walter J Chazin.

  Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.

Keywords:  DNA synthesis and repair; nucleotide excision repair; structural biology

DOI:  https://doi.org/10.1042/BST20210246
Elife. 2022 Jan 25. pii: e74218. [Epub ahead of print]11

Structure of the human ATM kinase and mechanism of Nbs1 binding.

Christopher Warren, Nikola P Pavletich.

  DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and cancer. Central to the sensing of DSBs is the ATM (Ataxia-telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. In response to DSBs, ATM is activated by the MRN (Mre11-Rad50-Nbs1) protein complex through a poorly-understood process that also requires double-stranded DNA. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM, and is required to retain active ATM at sites of DNA damage. Here we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning the N and C lobes along with relieving the blockage of the substrate-binding site. We also show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these structural findings with mutagenesis and biochemical assays.

Keywords:  human; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.74218
Nat Commun. 2022 Jan 25. 13(1): 501

USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma.

Yang Chen, Yin Zhao, Xiaojing Yang, Xianyue Ren, Shengyan Huang, Sha Gong, Xirong Tan, Junyan Li, Shiwei He, Yingqin Li, Xiaohong Hong, Qian Li, Cong Ding, Xueliang Fang, Jun Ma, Na Liu.

Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients.

DOI: https://doi.org/10.1038/s41467-022-28158-2
Clin Cancer Res. 2022 Jan 25. pii: clincanres.1846.2021. [Epub ahead of print]

A Novel Role for DNA-PK in Metabolism by Regulating Glycolysis in Castration Resistant Prostate Cancer.

Emanuela Dylgjeri, Vishal Kothari, Ayesha A Shafi, Galina Semenova, Peter T Gallagher, Yi Fang Guan, Angel Pang, Jonathan F Goodwin, Swati Irani, Jennifer J McCann, Amy C Mandigo, Saswati Chand, Christopher M McNair, Irina Vasilevskaya, Matthew J Schiewer, Costas D Lallas, Peter A McCue, Leonard G Gomella, Erin L Seifert, Jason S Carroll, Lisa M Butler, Jeff Holst, William K Kelly, Karen E Knudsen.

PURPOSE: DNA-dependent kinase catalytic subunit (DNA-PKcs, herein referred as DNA-PK) is a multifunctional kinase of high cancer relevance. DNA-PK is deregulated in multiple tumor types, including prostate cancer (PCa), and is associated with poor outcomes. DNA-PK was previously nominated as a therapeutic target and DNA-PK inhibitors are currently undergoing clinical investigation. While DNA-PK is well studied in DNA repair and transcriptional regulation, much remains to be understood about the way by which DNA-PK drives aggressive disease phenotypes.
EXPERIMENTAL DESIGN: Here, unbiased proteomic and metabolomic approaches in clinically relevant tumor models uncovered a novel role of DNA-PK in metabolic regulation of cancer progression. DNA-PK regulation of metabolism was interrogated using pharmacological and genetic perturbation using in vitro cell models, in vivo xenografts, and ex vivo in patient-derived explants (PDE).
RESULTS: Key findings reveal: i) the first-in-field DNA-PK protein-protein interactome; ii) numerous DNA-PK novel partners involved in glycolysis, iii) DNA-PK interacts with, phosphorylates (in vitro) and increases the enzymatic activity of glycolytic enzymes ALDOA and PKM2, iv) DNA-PK drives synthesis of glucose-derived pyruvate and lactate, v) DNA-PK regulates glycolysis in vitro, in vivo and ex vivo, and vi) combination of DNA-PK inhibitor with glycolytic inhibitor 2-deoxyglucose leads to additive anti-proliferative effects in aggressive disease.
CONCLUSIONS: Findings herein unveil novel DNA-PK partners, substrates, and function in PCa. The role of DNA-PK impacts glycolysis through direct interaction with glycolytic enzymes and modulation of enzymatic activity. These events support energy production that may contribute to generation and/or maintenance of DNA-PK-mediated aggressive disease phenotypes.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-1846
Cancer Chemother Pharmacol. 2022 Jan 23.

Dose-dependent bioavailability and tissue distribution of the ATR inhibitor AZD6738 (ceralasertib) in mice.

Brian F Kiesel, Jianxia Guo, Robert A Parise, Raman Venkataramanan, David A Clump, Christopher J Bakkenist, Jan H Beumer.

   PURPOSE: Ataxia telangiectasia and Rad3-related (ATR) initiates and regulates cellular responses to DNA damage, such as those caused by cancer treatments. Several ATR inhibitors (ATRi) are in clinical development including AZD6738. Therapeutic indices among ATRi may differ as a result of varying potencies and concentrations at both tumor and off-target sites. Additionally, AZD6738 contributes to anti-tumor immune responses necessitating evaluation of exposure at immunological sites.
METHODS: Using mouse models and a highly sensitive LC-MS/MS assay, the pharmacokinetics of AZD6738 were studied, including dose linearity, bioavailability, metabolism, and tissue distribution in tumor-bearing mice.
RESULTS: Initial studies identified dose-dependent bioavailability, with greater than proportional increases in exposure as dose increased resulting in a ~ twofold increase in bioavailability between the lowest and highest investigated doses. These behaviors were successfully captured with a compartmental PK model. Analysis of metabolite PK revealed decreasing metabolic ratios with increasing dose, indicative of saturable first-pass metabolism. Further analysis revealed that intestinal and gut metabolism contribute to metabolism and these saturable mechanisms. Studies of tumor and tissue distribution found rapid and extensive drug distribution to most tissues except brain and spinal cord.
CONCLUSION: The complex non-linear behavior of AZD6738 PK in mice was due to pre-systemic saturation and which appears to be recapitulated clinically at low doses. PK reported here will allow future correlation of tissue related toxicities with drug exposure as well as exposure with immunological responses. These results can also be compared with those from similar studies of other ATRi to contrast drug exposure with responses.

Keywords:  AZD6738; LC/MS; Pharmacokinetics; Small molecule inhibitor of ATR; Tissue distribution

DOI:  https://doi.org/10.1007/s00280-021-04388-x
Nat Commun. 2022 Jan 26. 13(1): 514

Mapping molecular subtype specific alterations in breast cancer brain metastases identifies clinically relevant vulnerabilities.

Nicola Cosgrove, Damir Varešlija, Stephen Keelan, Ashuvinee Elangovan, Jennifer M Atkinson, Sinéad Cocchiglia, Fiona T Bane, Vikrant Singh, Simon Furney, Chunling Hu, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Matthew P Goetz, Arnold D K Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie S Young.

The molecular events and transcriptional plasticity driving brain metastasis in clinically relevant breast tumor subtypes has not been determined. Here we comprehensively dissect genomic, transcriptomic and clinical data in patient-matched longitudinal tumor samples, and unravel distinct transcriptional programs enriched in brain metastasis. We report on subtype specific hub genes and functional processes, central to disease-affected networks in brain metastasis. Importantly, in luminal brain metastases we identify homologous recombination deficiency operative in transcriptomic and genomic data with recurrent breast mutational signatures A, F and K, associated with mismatch repair defects, TP53 mutations and homologous recombination deficiency (HRD) respectively. Utilizing PARP inhibition in patient-derived brain metastatic tumor explants we functionally validate HRD as a key vulnerability. Here, we demonstrate a functionally relevant HRD evident at genomic and transcriptomic levels pointing to genomic instability in breast cancer brain metastasis which is of potential translational significance.

DOI: https://doi.org/10.1038/s41467-022-27987-5
Mutagenesis. 2022 Jan 25. pii: geac003. [Epub ahead of print]

Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal.

Leigh Donnellan, Bradley S Simpson, Varinderpal S Dhillon, Maurizio Costabile, Michael Fenech, Permal Deo.

  Type 2 diabetes (T2D) is associated with elevated frequencies of micronuclei (MNi) and other DNA damage biomarkers. Interestingly, individuals with T2D are more likely to be deficient in micronutrients (folic acid, pyridoxal-phosphate, cobalamin) that play key roles in one-carbon metabolism and maintaining genomic integrity. Furthermore, it has recently been shown that deficiencies in these nutrients, in particular folic acid leaves cells susceptible to glucose-induced DNA damage. Therefore, we sought to investigate if the B lymphoblastoid WIL2-NS cell line cultured under folic acid-deficient conditions was more sensitive to DNA damage induced by glucose, or the reactive glycolytic byproduct methylglyoxal (MGO) and subsequent advanced glycation endproduct formation. Here, we show that only WIL2-NS cultured under folic acid-deficient conditions (23 nmol/l) experience an increase in MNi frequency when exposed to high concentrations of glucose (45 mmol/l) or MGO (100 µmol/l). Furthermore, we showed aminoguanidine, a well-validated MGO and free radical scavenger was able to prevent further MNi formation in folic acid-deficient cells exposed to high glucose, which may be due to a reduction in MGO-induced oxidative stress. Interestingly, we also observed an increase in MGO and other dicarbonyl stress biomarkers in folic acid-deficient cells, irrespective of glucose concentrations. Overall, our evidence shows that folic acid-deficient WIL2-NS cells are more susceptible to glucose and/or MGO-induced MNi formation. These results suggest that individuals with T2D experiencing hyperglycemia and folic acid deficiency may be at higher risk of chromosomal instability.

Keywords:  dicarbonyl stress; folic acid; glucose; micronuclei; nutrient interactions; oxidative stress

DOI:  https://doi.org/10.1093/mutage/geac003
Mol Cancer Ther. 2022 Jan 27. pii: molcanther.0604.2021. [Epub ahead of print]

Olaparib Induced Senescence is Bypassed through G2/M Checkpoint Override in Olaparib Resistant Prostate Cancer.

Alan P Lombard, Cameron M Armstrong, Leandro S D'Abronzo, Shu Ning, Amy R Leslie, Masuda Sharifi, Wei Lou, Christopher P Evans, Marc Dall'Era, Hong-Wu Chen, Xinbin Chen, Allen C Gao.

PARP inhibition represents the dawn of precision medicine for treating prostate cancer. Despite this advance, questions remain regarding the use of PARP inhibitors (PARPi's) for the treatment of this disease, including 1) how specifically do PARPi sensitive tumor cells respond to treatment, and 2) how does PARPi resistance develop? To address these questions, we characterized response to olaparib in sensitive LNCaP and C4-2B cells and developed two olaparib resistant derivative cell line models from each, termed LN-OlapR and 2B-OlapR respectively. OlapR cells possess distinct morphology from parental cells and display robust resistance to olaparib and other clinically relevant PARPi's including rucaparib, niraparib, and talazoparib. In LNCaP and C4-2B cells, we found that olaparib induces massive DNA damage, leading to activation of the G2/M checkpoint, activation of p53, and cell cycle arrest. Furthermore, our data suggest that G2/M checkpoint activation leads to both cell death and senescence associated with p21 activity. In contrast, both LN-OlapR and 2B-OlapR cells do not arrest at G2/M and display a markedly blunted response to olaparib treatment. Interestingly, both OlapR cell lines harbor increased DNA damage relative to parental cells, suggesting that OlapR cells accumulate and manage persistent DNA damage during acquisition of resistance, likely through augmenting DNA repair capacity. Further impairing DNA repair through CDK1 inhibition enhances DNA damage, induces cell death, and sensitizes OlapR cells to olaparib treatment. Our data together further our understanding of PARPi treatment and provide a cellular platform system for the study of response and resistance to PARP inhibition.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0604
J Biol Chem. 2022 Jan 24. pii: S0021-9258(22)00078-3. [Epub ahead of print] 101638

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase.

Chia Wei Hsu, Mark L Sowers, Tuvshintugs Baljinnyam, Jason L Herring, Linda C Hackfeld, Hui Tang, Kangling Zhang, Lawrence C Sowers.

  The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase, hyTDG, which is comprised of a 29-amino acid sequence from human thymine DNA glycosylase linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hyTDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by gas chromatography-mass spectrometry (GC-MS/MS) quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

Keywords:  DNA damage; DNA repair; cytosine; deamination; glycosylase; mass spectrometry; mutation

DOI:  https://doi.org/10.1016/j.jbc.2022.101638
Mol Biol Cell. 2022 Jan 26. mbcE20070433

Homology-directed repair involves multiple strand invasion cycles in fission yeast.

Amanda J Vines, Kenneth Cox, Bryan A Leland, Megan C King.

Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). How homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair often involves multiple site-specific and Rad51-dependent co-localization events between the DSB and donor sequence. Upon loss of the RecQ helicase Rqh1 (BLM in humans) we observe rapid repair possibly involving a single strand invasion event, suggesting that multiple strand invasion cycles antagonized by Rqh1 could reflect ongoing SDSA. However, failure to colocalize with the donor sequence and execute repair is also more likely in rqh1Δ cells, possibly reflecting erroneous strand invasion. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

DOI: https://doi.org/10.1091/mbc.E20-07-0433
PLoS Pathog. 2022 Jan;18(1): e1010210

Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools.

Rosario Sabariegos, Ana María Ortega-Prieto, Luis Díaz-Martínez, Ana Grande-Pérez, Carlos García Crespo, Isabel Gallego, Ana I de Ávila, Laura Albentosa-González, María Eugenia Soria, Pablo Gastaminza, Esteban Domingo, Celia Perales, Antonio Mas.

In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua, and not the other standard nucleosides, inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphates (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTPs or NDPs modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.

DOI: https://doi.org/10.1371/journal.ppat.1010210
Cancer Res. 2022 Jan 25. pii: canres.0218.2021. [Epub ahead of print]

Targeting cellular iron homeostasis with ironomycin in diffuse large B cell lymphoma.

Julie Devin, Tatiana Cañeque, Yea-Lih Lin, Lucie Mondoulet, Jean-Luc Veyrune, Matthieu Abouladze, Elvira Garcia De Paco, Ouissem Karmous Gadacha, Guillaume Cartron, Philippe Pasero, Caroline Bret, Raphaël Rodriguez, Jerome Moreaux.

Diffuse large B cell lymphoma (DLBCL) is the most common hematological malignancy. Although more than half of DLBCL patients achieve long-term remission, the majority of remaining patients succumb to the disease. As abnormal iron homeostasis is implicated in carcinogenesis and the progression of many tumors, we searched for alterations in iron metabolism in DLBCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of large cohorts of DLBCL patients established the Iron Score (IS), a gene expression-based risk score enabling identification of DLBCL patients with a poor outcome who might benefit from a suitable targeted therapy. In a panel of 16 DLBCL cell lines, ironomycin, a promising lysosomal iron-targeting small molecule, inhibited DLBCL cell proliferation at nanomolar concentrations compared to typical iron chelators. Ironomycin also induced significant cell growth inhibition, ferroptosis, and autophagy. Ironomycin treatment resulted in accumulation of DNA double strand breaks, delayed progression of replication forks, and increased RPA2 phosphorylation, a marker of replication stress. Ironomycin significantly reduced the median number of viable primary DLBCL cells of patients without major toxicity for non-tumor cells from the microenvironment and presented low toxicity in hematopoietic progenitors compared to conventional treatments. Significant synergistic effects were also observed by combining ironomycin with Doxorubicin, BH3 mimetics, BTK inhibitors, or Syk inhibitors. Altogether, these data demonstrate that a subgroup of high-risk DLBCL patients can be identified with the IS that can potentially benefit from targeting iron homeostasis.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0218
J Biol Chem. 2022 Jan 24. pii: S0021-9258(22)00075-8. [Epub ahead of print] 101635

Elimination of Aicardi Goutières Syndrome Protein SAMHD1 Activates Cellular Innate Immunity and Suppresses SARS-CoV-2 Replication.

Adrian Oo, Keivan Zandi, Caitlin Shepard, Leda C Bassit, Katie Musall, Shu Ling Goh, Young-Jae Cho, Dong-Hyun Kim, Raymond F Schinazi, Baek Kim.

  The lack of antiviral innate immune responses during SARS-CoV-2 infections are characterized by limited production of interferon proteins (IFNs). One protein associated with Aicardi Goutières syndrome, sterile alpha motif and HD-domain-containing protein 1 (SAMHD1), has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and HCoV-OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR/Cas9 gene knockout (KO) or lentiviral Vpx-mediated proteosomal degradation. We show that both SARS-CoV-2 and HCoV-OC43 replication were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by Jak inhibitor baricitinib, which overrode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2 infected patients primarily at the post-viral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.

Keywords:  JAK pathway; SAMHD1; SARS-CoV-2; Stat1; innate immunity

DOI:  https://doi.org/10.1016/j.jbc.2022.101635