Revolutionizing DNA repair research and cancer therapy with CRISPR–Cas screens

Nature Reviews Molecular Cell Biology, Год журнала: 2023, Номер 24(7), С. 477 - 494

Опубликована: Фев. 13, 2023

Язык: Английский

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PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities

Science Advances, Год журнала: 2023, Номер 9(37)

Опубликована: Сен. 13, 2023

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little known about ADP-ribosylation activity PARP14, including its substrate specificity or how PARP14-dependent reversed. We show that dual-function enzyme with both ADP-ribosyl hydrolase acting on protein nucleic acid substrates. In particular, we macrodomain 1 an active hydrolase. also demonstrate hydrolytic for first PARP9. reveal expression mutant inactivated results marked increase mono(ADP-ribosyl)ation proteins human cells, itself antiviral PARP13, displays specific cellular phenotypes. Moreover, closely related hydrolytically SARS2 Nsp3, Mac1, efficiently reverses vitro supporting evolution viral macrodomains to counteract PARP14-mediated response.

Язык: Английский

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Freedom to err: The expanding cellular functions of translesion DNA polymerases

Molecular Cell, Год журнала: 2023, Номер 83(20), С. 3608 - 3621

Опубликована: Авг. 24, 2023

Язык: Английский

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Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES

Chemical Research in Toxicology, Год журнала: 2024, Номер 37(2), С. 199 - 207

Опубликована: Янв. 10, 2024

Recent studies have defined a novel pathway for the repair of interstrand cross-links derived from reaction an adenine residue with apurinic/apyrimidinic (AP) site on opposing strand DNA (dA-AP ICL). Stalling replication fork at dA-AP ICL triggers TRAIP-dependent ubiquitylation CMG helicase that recruits base excision glycosylase NEIL3 to lesion. unhooks regenerate native one and AP other strand. Covalent capture abasic by SRAP protein HMCES protects against genomic instability would result cleavage in context single-stranded fork. After synthesis moves HMCES-AP adduct into double-stranded DNA, DNA–protein cross-link is resolved nonproteolytic mechanism involving dissociation thiazolidine attachment. The duplex then repaired pathway.

Язык: Английский

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FIRRM cooperates with FIGNL1 to promote RAD51 disassembly during DNA repair

Science Advances, Год журнала: 2023, Номер 9(32)

Опубликована: Авг. 9, 2023

Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors resolution ICL-induced double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator recombination and mitosis (FIRRM) as a sensitizer ICL-inducing agent mafosfamide. Mechanistically, showed FIRRM, like its interactor Fidgetin 1 (FIGNL1), contributes to RAD51 foci at DSBs. While stability FIRRM is interdependent, expression mutant (∆WCF), which stabilizes protein absence FIGNL1, allows cell survival, suggesting has FIGNL1-independent function during repair. In line with this model, binds preferentially single-stranded vitro, raising possibility it directly disassembly by DNA. Together, our findings establish promoting factor

Язык: Английский

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SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

EMBO Reports, Год журнала: 2022, Номер 23(4)

Опубликована: Фев. 14, 2022

Article14 February 2022Open Access Transparent process SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway Lisa Schubert Protein Signaling Program, Novo Nordisk Foundation Center for Research, University Copenhagen, Denmark Contribution: Conceptualization, Data curation, Funding acquisition, ​Investigation, Methodology, Project administration Search more papers by this author Ivo A Hendriks orcid.org/0000-0002-1439-3701 Proteomics Methodology Emil P T Hertz Formal analysis, Wei Wu Chromosome Stability, Department Cellular and Molecular Medicine, ​Investigation Selene Sellés-Baiget Saskia Hoffmann Keerthana Stine Viswalingam orcid.org/0000-0002-2179-1804 Biology, Irene Gallina orcid.org/0000-0002-3741-9038 Satyakrishna Pentakota Bente Benedict orcid.org/0000-0002-7503-8527 Joachim Johansen Disease Systems Katja Apelt Human Genetics, Leiden Medical Center, Leiden, The Netherlands Martijn S Luijsterburg Supervision Simon Rasmussen orcid.org/0000-0001-6323-9041 Michael Lisby orcid.org/0000-0002-4830-5247 Supervision, Writing - original draft, administration, review & editing Ying Liu L Nielsen Niels Mailand Corresponding Author [email protected] orcid.org/0000-0002-6623-709X Julien Duxin orcid.org/0000-0001-9389-4186 Information Schubert1, Hendriks2,†, Hertz1,†, Wu3,†, Sellés-Baiget1, Hoffmann1, Viswalingam4, Gallina1, Pentakota1, Benedict1, Johansen5, Apelt6, Luijsterburg6, Rasmussen5, Lisby3,4, Liu3, Nielsen2, *,1,3 *,1 1Protein 2Proteomics 3Center 4Department 5Disease 6Department † These authors contributed equally to work *Corresponding author. Tel: +45 35325023; E-mail: ***Corresponding 93565571; EMBO Reports (2022)23:e53639https://doi.org/10.15252/embr.202153639 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract (ICLs) are cytotoxic lesions that threaten genome integrity. (FA) orchestrates ICL during replication, with ubiquitylated FANCI-FANCD2 (ID2) marking activation step triggers incisions on unhook ICL. Restoration intact requires coordinated actions polymerase ζ (Polζ)-mediated translesion synthesis (TLS) homologous recombination (HR). While proteins mediating FA have been well characterized, effectors regulating choice promote resolution remain poorly defined. Here, we uncover an indispensable role in ensuring upon pathway. We show forms complex Polζ localizes ICLs replication. SCAI-deficient cells exquisitely sensitive ICL-inducing drugs display major hallmarks gene inactivation. In absence SCAI, HR-mediated is defective, breaks instead re-ligated θ-dependent microhomology-mediated end-joining, generating deletions spanning site radial chromosomes. Our establishes as integral component, acting at interface between TLS HR repair. Synopsis This critical regulator crosslink (ICL) Loss manifests deficiency ensures faithful replication-coupled interacts direct binding REV3 prevents end joining Introduction pathways counteract wide spectrum protect from detrimental mutations, breaks, chromosome rearrangements. some directly removed dedicated enzymes (e.g., oxidized bases readily restored base excision repair), other deleterious such require precise coordination multiple enzymatic activities re-establish DNA. highly lesions, whose tightly coupled replication (Akkari et al, 2000; Räschle 2008). large number participate repair, defects least 22 which causative (FA), rare, inherited disease characterized developmental birth defects, bone marrow failure, cancer predisposition (Rageul Kim, 2020). factors can be functionally subdivided according where they operate multistep (Wang, 2007; Ceccaldi 2016; Rageul First, core (comprising FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FANCM, FAAP20, FAAP100) associates damaged chromatin monoubiquitylates heterodimer UBE2T (FANCT) (Garcia-Higuera 2001; Smogorzewska Hira 2015; Rickman 2015). Monoubiquitylated ID2 marks downstream effector (Knipscheer 2009; Douwel 2014). include nucleases nick one strand both sides it (SLX4 (FANCP)-XPF (FANCQ)-ERCC1 complex), bypasses adduct (REV1-Polζ), (HR) two-ended double-strand break (DSB) final DSB distinct processes, prior restoration template TLS. Thus, must ensure but these processes largely unknown. demonstrate protein (suppressor cell invasion) has pathway, operating steps prevent erroneous intermediates Polθ-dependent (MMEJ), thereby resolution. Results principal To identify novel involved carried out genome-scale CRISPR-Cas9 dropout screen genes required survival presence low dose drug mitomycin C (MMC), corresponding 20% lethality (LD20). end, human RPE-1 targeted knockout (KO) p53 were transfected lentiviral sgRNA library propagated or MMC 12 days (Fig 1A). Validating our screening approach, many FA-associated scored among top hits (Figs 1B EV1A; Dataset EV1). fact, KO 20 known manifested hypersensitivity 1B). Unexpectedly, also revealed sgRNAs targeting selectively depleted exposed 1B; was originally identified transcriptional suppressor migration (Brandt 2009) later found play interaction 53BP1 (Hansen Isobe 2017). However, unlike did not significant MMC-treated (Dataset thus suggested previously unrecognized function set further explore. accordance screen, cisplatin, another inducer, could fully rescued stable re-expression 1C–E EV1B C). As observed established genes, displayed strong accumulation G2 phase accompanied increased level damage foci following treatment 1F–H EV1D–H). Likewise, metaphase spreads exhibited marked increase breaks/gaps formation treatment, characteristic feature patient 1I–K; García-de-Teresa Again, all phenotypes complemented ectopic 1F–K). conclude loss hypersensitizes agents phenocopies defective resulting Figure 1. dysfunction Schematic outline sensitizes MMC. LD20, lethal dose; NGS, next-generation sequencing. DrugZ analysis depletion CRISPR (A) low-dose (n = 2 technical replicates). highlighted blue; red. Immunoblot U2OS WT, U2OS/SCAI KO, stably reconstituted Strep-HA-SCAI (U2OS/SCAI KO/Strep-HA-SCAI). Clonogenic KO/Strep-HA-SCAI subjected indicated doses 24 h (mean ± SEM; n 3 independent experiments). (D), except treated Cisplatin (9 nM) 48 h, fixed, co-stained PCNA antibody DAPI. Cell cycle distribution analyzed quantitative image-based cytometry (QIBC) (≥ 2,000 per condition). representative experiment shown. (90 1 fixed later, RPA2 quantified QIBC 3,000 condition; mean SD; experiments; *P < 0.05; ns, significant, two-tailed paired t-test). (G), γH2AX DAPI **P 0.01; Experimental workflow morphology (top) images lines (bottom). stained Scale bars, 10 µm. Quantification chromosomes (I) 180 0.01, chromosomal 99 each condition pooled three ****P 0.0001, Mann–Whitney U test). Download figure PowerPoint Click here expand figure. EV1. (related Figs 2) GO term (NormZ −3) Fig 1A, using Reactome PANTHER16.0. WT (B), cisplatin Scatter plot showing 1F. Light grey, G1 phase; dark red: G2/M phase. Proportion indicated. (E), RAD51 Representative experiments 1G H. bar, (E) (F). FANCD2 siRNA knockdown efficiency cells. non-targeting control (CTRL) siRNAs harvested times after exposure (0.5 µM). Preventing alleviates better define emerging complementary suppresses aim, transduced near-lethal (LD80) 2A). Strikingly, ontology (GO) components most enriched class inactivation conferred significantly resistance 2B comprised associated (FANCA, FAAP100), complex, its deubiquitylation (USP1 WDR48), ordinarily roles activating against toxicity C; EV2; By contrast, functioning ubiquitylation score EV2). Genes encoding remodeling histone acetylation regulators proliferation far less prominently represented than 2C; Verifying results, FANCA suppressed DSBs 2D–G). Knockdown similarly alleviated EV1I J). Notably, much stronger background 2D E, exclude possibility consequence residual due incomplete siRNAs, knocked 2H). confirmed indeed cells, strongly 2I Importantly, whereas monoubiquitylation abolished expected, had no impact 2K EV1K). Collectively, data suggest essential proper once their processing initiated, se. 2. LD80, 80% MaGECK enrichment replicates; false discovery rate (FDR) 0.2 dotted line). blue. (FDR 0.2) (A), (F), lines. U2OS/FANCA U2OS, U2OS/FANCA+SCAI double (DKO) DKO elucidate how functions used Xenopus egg extracts, efficiently recapitulate plasmid containing site-specific (pICLpt) (Räschle system, two replisomes quickly converge lesion 3A, i). Upon collision, CMG first TRAIP unloaded p97 ii) (Fullbright 2019). One leading strands then extended within nucleotide (nt) (−1 position) 2008), point forks undergoes reversal produce substrate suitable iii) (Amunugama 2018). Ubiquitylated SLX4-XPF-ERCC1 iv), daughter molecules while leaving gap adducted molecule two-step involves insertion across unknown polymerase, extension past REV1-Polζ v) (Budzowska Finally, repaired utilizing vi–vii; Long 2011). 3. Current model ICLpt extracts pICLpt isolated pull down incubation extract CDC7i (100 µM) immunoblotting. replicated [α-32P]dATP times, reactions native agarose gel electrophoresis. inhibitor NMS-873 (p97i; 200 μM) supplemented reaction Note activity, CMGs longer plasmid, intermediate products (RI) 2016). OC, open circular; SC, supercoiled. Samples

Язык: Английский

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Catalytic and noncatalytic functions of DNA polymerase κ in translesion DNA synthesis

Nature Structural & Molecular Biology, Год журнала: 2024, Номер unknown

Опубликована: Сен. 19, 2024

Язык: Английский

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Fanconi anemia-associated chromosomal radial formation is dependent on POLθ-mediated alternative end joining

Cell Reports, Год журнала: 2023, Номер 42(5), С. 112428 - 112428

Опубликована: Апрель 21, 2023

Activation of the Fanconi anemia (FA) pathway after treatment with mitomycin C (MMC) is essential for preventing chromosome translocations termed "radials." When replication forks stall at MMC-induced interstrand crosslinks (ICLs), FA activated to orchestrate ICL unhooking and repair DNA break intermediates. However, in FA-deficient cells, how ICL-associated breaks are resolved a manner that leads radials unclear. Here, we demonstrate dependent on polymerase theta (POLθ)-mediated alternative end joining (A-EJ). Specifically, show observed FANCD2−/− cells POLθ ligase III occur independently classical non-homologous joining. Furthermore, inhibitors abolishes accumulation co-localizing common fragile sites. Uniformly, these observations implicate A-EJ radial formation provide mechanistic insights into pathway-deficient cancers inhibitors.

Язык: Английский

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Research progress of the Fanconi anemia pathway and premature ovarian insufficiency

Biology of Reproduction, Год журнала: 2023, Номер 109(5), С. 570 - 585

Опубликована: Сен. 5, 2023

Abstract The Fanconi anemia pathway is a key involved in the repair of deoxyribonucleic acidinterstrand crosslinking damage, which chiefly includes following four modules: lesion recognition, core complex recruitment, FANCD2–FANCI monoubiquitination, and downstream events (nucleolytic incision, translesion synthesis, homologous recombination). Mutations or deletions multiple genes this can damage interstrand disrupt primordial germ cell development oocyte meiosis, thereby leading to abnormal follicular development. Premature ovarian insufficiency gynecological clinical syndrome characterized by amenorrhea decreased fertility due pool, accelerated follicle atresia, loss function women &lt;40 years old. Furthermore, recent years, several studies have detected mutations gene patients with premature insufficiency. In addition, some exhibit symptoms infertility. are closely associated.

Язык: Английский

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Repair of genomic interstrand crosslinks

DNA repair, Год журнала: 2024, Номер 141, С. 103739 - 103739

Опубликована: Июль 30, 2024

Язык: Английский

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