Timeless couples G‐quadruplex detection with processing by DDX 11 helicase during DNA replication

• DOI: 10.15252/embj.2019104185
• Publication Date: 2020-07-24T06:58:35Z
• Authors: Leticia K Lerner; Sandro Holzer; Mairi L Kilkenny; Saša Šviković; Pierre Murat; Davide Schiavone; Cara B Eldridge; Alice Bittleston; Joseph D Maman; Dana Branzei; Katherine Stott; Luca Pellegrini; Julian E Sale
• Keywords: DNA Replication; 0303 health sciences; G-quadruplex; replisome; fork protection complex; timeless; DNA Helicases; Intracellular Signaling Peptides and Proteins; Cell Cycle Proteins; Articles; DNA replication; Cell Line; DEAD-box RNA Helicases; G-Quadruplexes; 03 medical and health sciences; Protein Domains; [CHIM] Chemical Sciences; [CHIM]Chemical Sciences; Humans; Recombination & Repair; DNA Damage

Article23 July 2020Open Access Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication Leticia K Lerner MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers this author Sandro Holzer Department Biochemistry, University Mairi L Kilkenny Saša Šviković Pierre Murat Davide Schiavone Cara B Eldridge Alice Bittleston Joseph D Maman Dana Branzei orcid.org/0000-0002-0544-4888 IFOM, Fondazione Italiana per la Ricerca sul Cancro, Institute Oncology, Milan, Italy Katherine Stott Luca Pellegrini Corresponding Author [email protected] orcid.org/0000-0002-9300-497X Julian E Sale orcid.org/0000-0002-5031-3780 Information Lerner1,4,‡, Holzer2,‡, Kilkenny2, Šviković1, Murat1, Schiavone1, Eldridge1, Bittleston2, Maman2, Branzei3, Stott2, *,2 and *,1 1MRC 2Department 3IFOM, 4Present address: Centre de Recherche des Cordeliers, Cell Death Drug Resistance in Hematological Disorders Team, INSERM UMRS 1138, Sorbonne Université, Paris, France ‡These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: 267099; The EMBO Journal (2020)39:e104185https://doi.org/10.15252/embj.2019104185 See also: CH Freudenreich (September 2020) PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision process including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Regions genome potential form secondary structures pose frequent significant impediment must be actively managed order preserve genetic epigenetic integrity. How replisome detects responds is poorly understood. Here, we show that core component fork protection complex eukaryotic replisome, Timeless, harbours its C-terminal region previously unappreciated DNA-binding domain exhibits specific binding (G4) structures. We contributes maintaining processive through G4-forming sequences, partial redundancy an adjacent PARP-binding domain. Further, function requires interaction activity DDX11. Loss both causes instability at sequences damage. Our findings indicate ability sense replication-hindering G4 formation ensures prompt resolution these maintain synthesis. Synopsis replisomes detect respond cooperation subunit found bind resolve replication. contains specificity acts G4s. G4s helicase. or leads G4-associated damage Introduction can create impediments own When unwound, certain often repetitive low complexity, adopt variety non-B structures, hairpins, cruciforms, triplexes quadruplexes (Mirkin Mirkin, 2007). It increasingly clear structure event replication, even genomically abundant thought not major source difficulty (Šviković et al, 2019). To prevent such causing havoc stability genome, cells deploy intricate network activities counteract limit effects. These include proteins destabilise specialised helicases unwind them (Lerner Sale, In addition, repriming PrimPol deployed confine into minimal single-stranded (ssDNA), limiting dangers exposing extensive ssDNA stalled (Schiavone 2016; are one most intensively studied potent structural impediments. arise consequence guanine Hoogsteen base-paired quartets (Gellert 1962). favourable sequence contexts, comprising runs dG separated variable numbers non-G bases, stacks G Current estimates suggest over 700,000 sites human have (Chambers 2015). While some may important roles physiology, all threat been linked 2017; Kaushal Freudenreich, Precisely, how detected resolved machinery remains unclear. Many factors involved instance FANCJ REV1 (Kruisselbrink 2008; London Wu Youds Sarkies 2010), do appear constitutive components (Dungrawala thus likely will act as "first responders" play role coupling their suppressing deleterious effects on Particularly interesting context subset known (FPC). FPC comprises four proteins—Timeless, Tipin, Claspin AND-1—that conserved from yeast mammals (Errico Costanzo, 2012). associate via direct interactions CMG replicative polymerases α, δ ε (Nedelcheva 2005; Numata 2010; Cho 2013; Bastia Baretić 2020). They also interact directly (Tanaka 2010) indirectly protein A (RPA) (Witosch 2014). allow remain fork, which normal speed synthesis (Yeeles 2017). Additionally, has series functions promote progression integrity: it essential avoid uncoupling pol consequent long stretches (Katou 2003; Lou 2008). S-phase checkpoint activation response damage, kinase activation, cell cycle arrest maintenance integrity (Chou Elledge, 2006; Gotter 2007; Unsal-Kaçmaz Yang 2010). Furthermore, plays important, but incompletely understood, sister chromosome cohesion (Chan Leman well placed metabolism could impede Indeed, deficiency TOF1 stalling, repeat fragility structure-forming (Voineagu 2008, 2009; 2012; Liu 2012b; Gellon 2019), underscoring importance regions capable forming Although itself does possess catalytic would interacts (Calì 2016). (or CHLR1), 5′–3′ Fe–S same superfamily 2 FANCJ, RTEL XPD humans, mutations cause Warsaw breakage syndrome, extremely rare autosomal recessive disease characterised microcephaly, growth retardation, cochlear abnormalities abnormal skin pigmentation (Alkhunaizi 2018). vitro, unwinding several non-duplex (Wu 2012a; Bharti 2013), triplex (Guo 2015) D-loops 2012a). enhanced However, unclear collaborate vivo detecting provide evidence operate together ensure DNA. report (DBD) C-terminus towards propose recruiting maintained, thereby avoiding G4-induced instability. Results required genomic motif address whether vivo, disrupted TIMELESS locus chicken DT40 CRISPR/Cas9-induced deletions. isolated timeless mutants biallelic disruptions exon 1, around guide site (Appendix Fig S1). mutant were sensitive cisplatin (Fig EV1), observed depleted (Liu assess sequence, took advantage Bu-1 loss variant assay stable expression BU-1 dependent located ~ 3.5 kb downstream promoter 11A). Prolonged pausing leading-strand information gene permanent heritable change (Sarkies 2014, Guilbaud This stochastic replication-dependent generation variants monitored flow cytometry encodes surface glycoprotein. Small pools Bu-1high wild-type expanded parallel 20 divisions (15–21 days), proportion each pool had lost status determined. increased levels compared cells, retained 11B C). was fully reversed 11C) +3.5 11C). Cells deficient Tipin (Abe 2016), interactor within FPC, exhibit 11D). results necessary motif. Click here expand figure. Figure EV1. Sensitivity wild type (WT), timeless, ddx11 fancj (CDDP)Cell viability, assessed MTS assay, type, ddx11, fancj, after 72 h presence indicated doses. values represent means (error bars SD) two independent experiments performed triplicate. *P < 0.05, ***P 0.001 ****P 0.0001; one-way ANOVA type. Download figure PowerPoint 1. past model system record G4-dependent stalling. leading strand entering 3′ end stochastically stalls G4, ssDNA, interruption parental histone recycling modifications Instability cells. FACS plots (clone 1) stained anti-Bu-1 conjugated phycoerythrin. Each line represents profile individual clonal population. Unstained controls shown blue. Fluctuation analysis clones generated CRISPR-Cas9 targeting (clones 1 2; Appendix S1), complemented cDNA background endogenous deleted (ΔG4) tipin Data information: (C) (D), symbol percentage clone 2–3 weeks expression. At least fluctuation analyses performed, 24–36 repeat. Bars whiskers median interquartile range, respectively. ANOVA. Identification characterisation As intimately associated vitro data Timeless–Tipin RPA 2014), Swi1-Swi3 complex, fission orthologue Timeless–Tipin, Inspection amino acid revealed half (residues 816–954; 22A), predicted fold similarity myb-like homeodomain-like superfamily, double-stranded (dsDNA) tandem 3-helix bundles (named N-term C-term). 2. Schematic drawing (NTD: N-terminal domain; DBD: PBD: domain). multiple alignment vertebrate underneath, conservation coloured according Clustal colour scheme. annotated extent elements helical domains (N-term C-term) composing DBD. Ribbon 1.15 Å crystal DBD C-term. Helices red labelled H1–H4. drawings C-term determined NMR. orientation highlight high degree three-dimensional similarity. superposition lowest energy affinity measured fluorescence anisotropy, titrating against Cy3 3′-labelled dsDNA (see Table S2 details). top panel shows curves ss- dsDNA, bottom curve substrate. points mean three experiments, error standard deviation (SD). diagram highly similar telomeric TRF1 (PDB ID 1W0T) (Court 2005) bacterial regulator GcrA 5Z7I) 2018) substrates. mode steric overlap helix H4 phosphate backbone dsDNA. light blue, brown substrates khaki. used X-ray diffraction NMR spectroscopy investigate experimentally dynamics newly discovered acids 885–947 (C-term), corresponding single fold, confirmed three-helix bundle characteristic homeodomain proteins, extended fourth alpha unique 22B). ensemble 816–954 well-converged (824–880 891–944, r.m.s.d. 0.4 0.5 Å, respectively) connected linker (881–890) significantly less converged, implying flexibility between N- 22C). observation measurements S2). adopted fold; particular, shared additional helix, H4, seen keeping (DBDs), examined bound micromolar probes 22D; panel). distinguishing feature repeats. Superposition onto structurally homologous 22E) that, if binding, repeats lead clash conceivable might rearrange conformation upon participates hydrophobic interactions, mediated residues L872 F879 (N-terminal repeat), L936, V937 L943 (C-terminal making rearrangements unlikely. Given functional operates observations, presented 11, speculated directed recognition transiently unwound template, when tested well-characterised present MYC (Ambrus 2005), nanomolar affinity, about magnitude tighter than ds- 22D). prompted us ask like DBD, able mimic template DNA, embedded longer (ssG4; ssG4 selective fashion, did measurable hairpin (ssHP), mutated (ss; Figs 33A EV2, next different contexts folding topologies: them, albeit affinities varied whereas appreciable 33B). 3. preference DNAFluorescence anisotropy measure sequences. ssG4: flanked DNA; ssHP: ss: Binding range details references). Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) (ds20: ss20 annealed complementary sequence) controls. point 3 SD. EV2. Coomassie-stained SDS–PAGE gel purified complex. Electrophoretic mobility shift (EMSA) showing BU1A + 3.5. Mutation (BU1A mut) disrupts final concentration 5 μM. EMSA (ssG4, dsG4) (ss), (ds) hairpin-containing (ssHP, dsHP). contains, half, unrecognised closely resembles transcription factors. binds 10-fold greater defined sequence. crucial further explore contribution expressing version truncating before using CRISPR/Cas9 16. truncation removes reported PARP1, PARP1-binding (PBD) (Xie exhibited comparable 44) suggesting result truncated 816, lacking PBD, S3). dissect observation, versions only (ΔDBD) PBD (PARP*). either largely restored, although completely, redundantly. co-immunoprecipitation (Co-IP) showed neither EV3), partner whose stimulated PDB contribute, independently recruitment. 4. replicationFluctuation variants. Top bottom: 1), (timeless ∆C) 16 truncates removing CTD containing domains. Then, complementation timeless#1 (hTim), hTim∆816–1,208 (lacking PBD), hTim∆816–965 DBD) hTim[1:1,000], PBD. 0.05 comparison EV3. DDX11HEK293T transfected plasmid encoding Flag-hTimeless, co-transfected plasmids hDDX11 Flag-Timeless delete (ΔDBD: deletion S816–S965) (PARP*: V1000). Twenty-four transfection, whole-cell extracts subjected immunoprecipitation anti-Flag magnetic beads. Western Blot overexpressed pulled down samples antibody. Upper panel: Input constructs Bottom anti-DDX11 Tubulin loading control input samples. Since lacks any activity, explored accounted system. noted above, (Leman Calì Cortone 2018), chromatid (Cortone preservation perturbed condi