Timeless couples G‐quadruplex detection with processing by DDX 11 helicase during DNA replication
2020; Springer Nature; Volume: 39; Issue: 18 Linguagem: Inglês
10.15252/embj.2019104185
ISSN1460-2075
AutoresLetícia Koch Lerner, Sandro Holzer, M.L. Kilkenny, Saša Šviković, Pierre Murat, Davide Schiavone, Cara Bernadette Eldridge, Alice Bittleston, Joseph D. Maman, Dana Branzei, Katherine Stott, Luca Pellegrini, Julian E. Sale,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle23 July 2020Open Access Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication Leticia K Lerner Leticia K Lerner MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sandro Holzer Sandro Holzer Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Mairi L Kilkenny Mairi L Kilkenny Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Saša Šviković Saša Šviković MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Pierre Murat Pierre Murat MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Davide Schiavone Davide Schiavone MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Cara B Eldridge Cara B Eldridge MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Alice Bittleston Alice Bittleston Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Joseph D Maman Joseph D Maman Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Dana Branzei Dana Branzei orcid.org/0000-0002-0544-4888 IFOM, Fondazione Italiana per la Ricerca sul Cancro, Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Katherine Stott Katherine Stott Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Luca Pellegrini Corresponding Author Luca Pellegrini [email protected] orcid.org/0000-0002-9300-497X Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Julian E Sale Corresponding Author Julian E Sale [email protected] orcid.org/0000-0002-5031-3780 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Leticia K Lerner Leticia K Lerner MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sandro Holzer Sandro Holzer Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Mairi L Kilkenny Mairi L Kilkenny Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Saša Šviković Saša Šviković MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Pierre Murat Pierre Murat MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Davide Schiavone Davide Schiavone MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Cara B Eldridge Cara B Eldridge MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Alice Bittleston Alice Bittleston Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Joseph D Maman Joseph D Maman Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Dana Branzei Dana Branzei orcid.org/0000-0002-0544-4888 IFOM, Fondazione Italiana per la Ricerca sul Cancro, Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Katherine Stott Katherine Stott Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Luca Pellegrini Corresponding Author Luca Pellegrini [email protected] orcid.org/0000-0002-9300-497X Department of Biochemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Julian E Sale Corresponding Author Julian E Sale [email protected] orcid.org/0000-0002-5031-3780 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Leticia K Lerner1,4,‡, Sandro Holzer2,‡, Mairi L Kilkenny2, Saša Šviković1, Pierre Murat1, Davide Schiavone1, Cara B Eldridge1, Alice Bittleston2, Joseph D Maman2, Dana Branzei3, Katherine Stott2, Luca Pellegrini *,2 and Julian E Sale *,1 1MRC Laboratory of Molecular Biology, Cambridge, UK 2Department of Biochemistry, University of Cambridge, Cambridge, UK 3IFOM, Fondazione Italiana per la Ricerca sul Cancro, Institute of Molecular Oncology, Milan, Italy 4Present address: Centre de Recherche des Cordeliers, Cell Death and Drug Resistance in Hematological Disorders Team, INSERM UMRS 1138, Sorbonne Université, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1223 760469; E-mail: [email protected] *Corresponding author. Tel: +44 1223 267099; E-mail: [email protected] The EMBO Journal (2020)39:e104185https://doi.org/10.15252/embj.2019104185 See also: CH Freudenreich (September 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciated DNA-binding domain that exhibits specific binding to G-quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacent PARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4-forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis. Synopsis How eukaryotic replisomes detect and respond to DNA secondary structures is poorly understood. Here, cooperation of the fork protection complex subunit Timeless and the helicase DDX11 is found to bind and resolve G-quadruplex (G4) DNA during replication. Timeless, a core component of the replisome, contains a C-terminal DNA binding domain with specificity for G4 secondary structures. The DNA-binding domain acts in partial redundancy with an adjacent PARP-binding domain to maintain processive replication through G4s. The ability of Timeless to maintain replication through G4s requires interaction with the DDX11 helicase. Loss of Timeless or DDX11 leads to G4-associated epigenetic instability and DNA damage Introduction DNA can create significant impediments to its own replication through the formation of secondary structures. When unwound, certain sequences, often repetitive or of low complexity, can adopt a variety of non-B form structures, including hairpins, cruciforms, triplexes and quadruplexes (Mirkin & Mirkin, 2007). It is increasingly clear that secondary structure formation is a frequent event during replication, even at genomically abundant sequences previously thought not to be a major source of difficulty (Šviković et al, 2019). To prevent such sequences causing havoc with the genetic and epigenetic stability of the genome, cells deploy an intricate network of activities to counteract secondary structure formation and limit its effects. These activities include proteins that bind and destabilise DNA structures and specialised helicases that unwind them (Lerner & Sale, 2019). In addition, the repriming activity of PrimPol can be deployed to confine a structure into a minimal region of single-stranded DNA (ssDNA), limiting the potential dangers of exposing extensive ssDNA in a stalled replisome (Schiavone et al, 2016; Šviković et al, 2019). G4s are one of the most intensively studied and potent structural replication impediments. G4s arise in consequence of the ability of guanine to form Hoogsteen base-paired quartets (Gellert et al, 1962). In favourable sequence contexts, comprising runs of dG separated by variable numbers of non-G bases, stacks of G quartets form G4 secondary structures. Current estimates suggest that over 700,000 sites in the human genome have the potential to form G4s (Chambers et al, 2015). While some of these G4s may have important roles in genome physiology, all pose a potential threat to DNA replication and sites with G4-forming potential have been linked to both genetic and epigenetic instability (Šviković & Sale, 2017; Kaushal & Freudenreich, 2019). Precisely, how DNA structures are detected and resolved by the replication machinery remains unclear. Many of the factors involved in processing G4 secondary structures, for instance FANCJ and REV1 (Kruisselbrink et al, 2008; London et al, 2008; Wu et al, 2008; Youds et al, 2008; Sarkies et al, 2010), do not appear to be constitutive components of the replisome (Dungrawala et al, 2015). It is thus likely that core components of the replisome will act as "first responders" to DNA structures and play an important role coupling their detection with suppressing their deleterious effects on DNA synthesis. Particularly interesting in this context is a subset of replisome components known as the fork protection complex (FPC). The FPC comprises four main proteins—Timeless, Tipin, Claspin and AND-1—that are conserved from yeast to mammals (Errico & Costanzo, 2012). FPC components associate with the replication fork via direct interactions with the CMG replicative helicase and replicative polymerases α, δ and ε (Nedelcheva et al, 2005; Numata et al, 2010; Cho et al, 2013; Bastia et al, 2016; Kilkenny et al, 2017; Baretić et al, 2020). They also interact with DNA both directly (Tanaka et al, 2010) and indirectly via replication protein A (RPA) (Witosch et al, 2014). These interactions allow the FPC to remain at the fork, which ensures a normal speed of DNA synthesis (Yeeles et al, 2017). Additionally, the FPC has a series of functions that promote normal replisome progression and fork integrity: it is essential to avoid uncoupling of pol ε from the replicative helicase and consequent formation of long stretches of ssDNA (Katou et al, 2003; Lou et al, 2008). It also has a conserved role in S-phase checkpoint activation in response to DNA damage, including checkpoint kinase activation, cell cycle arrest and maintenance of the integrity of the replication fork (Chou & Elledge, 2006; Gotter et al, 2007; Unsal-Kaçmaz et al, 2007; Yang et al, 2010). Furthermore, it plays an important, but incompletely understood, role in maintaining sister chromosome cohesion (Chan et al, 2003; Leman et al, 2010). The FPC is thus well placed to play a role in the detection and metabolism of DNA secondary structures that could impede DNA synthesis. Indeed, deficiency of both TOF1 in yeast and Timeless in human cells leads to replication fork stalling, repeat instability and fragility at secondary structure-forming sequences (Voineagu et al, 2008, 2009; Leman et al, 2012; Liu et al, 2012b; Gellon et al, 2019), underscoring the potential importance of Timeless in maintaining processive replication through regions of the genome capable of forming secondary structures. Although Timeless itself does not appear to possess catalytic activity that would process DNA secondary structures, it interacts with the DNA helicase DDX11 (Calì et al, 2016). DDX11 (or CHLR1), is a 5′–3′ Fe–S helicase of the same superfamily 2 as FANCJ, RTEL and XPD (Lerner & Sale, 2019). In humans, mutations in DDX11 cause Warsaw breakage syndrome, an extremely rare autosomal recessive disease characterised by microcephaly, growth retardation, cochlear abnormalities and abnormal skin pigmentation (Alkhunaizi et al, 2018). In vitro, DDX11 has unwinding activity on several non-duplex DNA structures, such as G4s (Wu et al, 2012a; Bharti et al, 2013), triplex DNA (Guo et al, 2015) and D-loops (Wu et al, 2012a). Further, the helicase activity of DDX11 is enhanced by Timeless (Calì et al, 2016). However, it remains unclear how Timeless and DDX11 collaborate in vivo in detecting and processing G4s during replication. Here, we provide in vivo evidence that Timeless and DDX11 operate together to ensure processive replication of G4-forming DNA. We report a previously unappreciated DNA-binding domain (DBD) in the C-terminus of Timeless, which exhibits specificity towards G4 structures. We propose that Timeless plays a role in the detection of G4 structures at the replication fork, recruiting DDX11 to unwind them and ensure processive replication is maintained, thereby avoiding G4-induced genetic and epigenetic instability. Results Timeless is required for processive replication of a genomic G4 motif To address whether Timeless is involved in maintaining processive replication of G4-forming DNA in vivo, we disrupted the TIMELESS locus in chicken DT40 cells with CRISPR/Cas9-induced deletions. We isolated several timeless mutants with biallelic disruptions in exon 1, around the guide site (Appendix Fig S1). The timeless mutant cells were sensitive to cisplatin (Fig EV1), as previously observed in human cells depleted of Timeless (Liu et al, 2017). To assess the role of Timeless in the replication of a G4-forming sequence, we took advantage of the Bu-1 loss variant assay (Schiavone et al, 2014). The stable expression of the BU-1 locus in DT40 is dependent on the maintenance of processive replication through a G4 motif located ~ 3.5 kb downstream of the promoter (Fig 11A). Prolonged pausing of leading-strand replication at this motif leads to loss of epigenetic information around the promoter of the gene and a permanent and heritable change in its expression (Sarkies et al, 2012; Schiavone et al, 2014, 2016; Guilbaud et al, 2017). This stochastic and replication-dependent generation of Bu-1 loss variants can be monitored by flow cytometry as BU-1 encodes a surface glycoprotein. Small pools of Bu-1high wild-type and timeless cells were expanded in parallel for ~ 20 divisions (15–21 days), and the proportion of cells in each pool that had lost their Bu-1high status determined. We detected increased levels of Bu-1 expression instability in timeless DT40 cells compared to the wild-type cells, which retained their stable Bu-1high expression (Fig 11B and C). The instability of Bu-1 expression in timeless cells was fully reversed by expression of human Timeless (Fig 11C) and is dependent on the +3.5 G4 motif (Fig 11C). Cells deficient in Tipin (Abe et al, 2016), a constitutive interactor of Timeless within the FPC, also exhibit instability of BU-1 expression (Fig 11D). These results show that Timeless is necessary to maintain processive DNA replication of a genomic G4 motif. Click here to expand this figure. Figure EV1. Sensitivity of wild type (WT), timeless, ddx11 and fancj DT40 mutants to cisplatin (CDDP)Cell viability, assessed by MTS assay, of DT40 wild type, ddx11, timeless and fancj, after 72 h in presence of cisplatin at the indicated doses. The values represent the means (error bars indicate SD) of two independent experiments performed in triplicate. *P < 0.05, ***P < 0.001 and ****P < 0.0001; one-way ANOVA compared to the wild type. Download figure Download PowerPoint Figure 1. Timeless and Tipin are required to maintain processive replication past G4 structures in vivo The BU-1 locus as a model system to record G4-dependent replication stalling. The leading strand of a replication fork entering the locus from the 3′ end stochastically stalls at the +3.5 G4, leading to the formation of a region of ssDNA, with interruption of parental histone recycling and of histone modifications necessary to maintain normal expression of the locus (Schiavone et al, 2014). Instability of BU-1 expression in timeless cells. FACS plots of wild-type and timeless (clone 1) DT40 cells stained with anti-Bu-1 conjugated with phycoerythrin. Each line represents the Bu-1 expression profile of an individual clonal population. Unstained controls are shown in blue. Fluctuation analysis for Bu-1 loss in wild-type DT40 cells and two independent timeless clones generated by CRISPR-Cas9 targeting (clones 1 and 2; Appendix Fig S1), timeless (clone 1) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous +3.5 G4 has been deleted (ΔG4) (Schiavone et al, 2014). Fluctuation analysis for Bu-1 loss in DT40 wild-type and tipin cells. Data information: In (C) and (D), each symbol represents the percentage of cells in an individual clone expanded for 2–3 weeks that have lost Bu-1high expression. At least two independent fluctuation analyses were performed, with 24–36 individual clones each cell line per repeat. Bars and whiskers represent median and interquartile range, respectively. ****P < 0.0001; one-way ANOVA. Download figure Download PowerPoint Identification and characterisation of a Timeless DNA-binding domain As a core component of the replisome, Timeless is intimately associated with DNA synthesis at the replication fork (Yeeles et al, 2017). In vitro data show that the Timeless–Tipin complex can bind to ssDNA through RPA (Witosch et al, 2014), and the Swi1-Swi3 complex, the fission yeast orthologue of Timeless–Tipin, was also shown to bind DNA (Tanaka et al, 2010). Inspection of the amino acid sequence of human Timeless revealed the presence of a conserved domain in its C-terminal half (residues 816–954; Fig 22A), with a predicted fold similarity to the myb-like proteins of the homeodomain-like superfamily, that bind double-stranded DNA (dsDNA) with a tandem repeat of 3-helix bundles (named here N-term and C-term). Figure 2. Identification and characterisation of a DNA-binding activity in Timeless Schematic drawing of human Timeless and its known domain structure (NTD: N-terminal domain; DBD: DNA-binding domain; PBD: PARP-binding domain). A multiple sequence alignment of vertebrate Timeless sequences is shown underneath, with amino acid conservation coloured according to the Clustal colour scheme. The alignment is annotated with the extent and secondary structure elements of the two helical domains (N-term and C-term) composing the DBD. Ribbon drawing of the 1.15 Å crystal structure at of the DBD C-term. Helices are in red and labelled H1–H4. Ribbon drawings of the N-term and C-term domains of the DBD determined by NMR. The two domains are shown in the same orientation to highlight their high degree of three-dimensional similarity. The superposition of the 20 lowest energy structures is shown for each domain. The DNA-binding affinity of DBD was measured by fluorescence anisotropy, titrating the DBD protein against Cy3 3′-labelled ssDNA, dsDNA and G4 DNA (see Appendix Table S2 for sequence details). The top panel shows binding curves for ss- and dsDNA, and the bottom panel shows the binding curve for the G4 DNA substrate. The data points represent the mean of at least three independent experiments, and the error bars indicate one standard deviation (SD). Ribbon diagram of the superposition of DBD C-term with the highly similar DNA-binding domains of telomeric protein TRF1 (PDB ID 1W0T) (Court et al, 2005) and the bacterial cell cycle regulator GcrA (PDB ID 5Z7I) (Wu et al, 2018) in complex with their DNA substrates. A similar DNA-binding mode by DBD would cause a steric overlap of helix H4 with the phosphate backbone of dsDNA. DBD C-term is in light blue, TRF1 and GcrA proteins in brown and their DNA substrates in khaki. Download figure Download PowerPoint We used X-ray diffraction and NMR spectroscopy to investigate experimentally the structure and dynamics of the newly discovered Timeless domain. The 1.15 Å crystal structure of amino acids 885–947 (C-term), corresponding to a single myb-like fold, confirmed the presence of a three-helix bundle characteristic of the homeodomain superfamily of DNA-binding proteins, extended by the presence of a fourth C-terminal alpha helix unique to the Timeless domain (Fig 22B). The NMR structural ensemble of amino acids 816–954 revealed two well-converged domains (824–880 and 891–944, backbone r.m.s.d. 0.4 and 0.5 Å, respectively) connected by a linker (881–890) that was significantly less well converged, implying a high degree of flexibility between N- and C-term domains (Fig 22C). This observation was confirmed by backbone dynamics measurements (Appendix Fig S2). The N- and C-terminal domains adopted the same three-dimensional fold; in particular, the N-terminal repeat shared the presence of an additional fourth helix, H4, as seen in the C-terminal repeat (Fig 22C). In keeping with the similarity of its structure to known DNA binding domains (DBDs), we examined the ability of the Timeless domain to interact with DNA. We found that it bound with low micromolar affinity to both ss- and dsDNA probes (Fig 22D; top panel). A distinguishing feature of the Timeless DBD is the presence of a fourth alpha helix in both N- and C-terminal 3-helix bundle repeats. Superposition of the DBD C-term onto the structurally homologous domains of the telomeric protein TRF1 (Court et al, 2005) and bacterial cell cycle regulator GcrA (Wu et al, 2018) in complex with their dsDNA substrates (Fig 22E) shows that, if the DBD were to adopt a similar mode of dsDNA binding, the fourth helix of both its N-term and C-term repeats would likely lead to a steric clash with the phosphate backbone of the DNA. Although it is conceivable that the DBD might rearrange its conformation upon DNA binding, the fourth helix of both repeats participates in core hydrophobic interactions, mediated by conserved residues L872 and F879 (N-terminal repeat), and L936, V937 and L943 (C-terminal repeat), making such rearrangements unlikely. Given the functional context in which Timeless operates as a replisome component and the observations, presented in Fig 11, that it is required to maintain processive replication of the BU-1 G4, we speculated that the C-terminal DNA-binding activity of Timeless might be directed towards recognition of DNA secondary structures that form transiently on the unwound template, such as G4s. Indeed, when we tested a well-characterised G4 sequence present in the promoter of the MYC gene (Ambrus et al, 2005), the DBD bound to it with nanomolar affinity, and about one order of magnitude tighter than ds- or ssDNA (Fig 22D). This observation prompted us to ask whether the Timeless–Tipin complex, like the isolated DBD, is able to bind to the same G4 motif. To mimic the unwound template DNA, we embedded the G4 within a longer ssDNA sequence (ssG4; Appendix Table S2). We found that the Timeless–Tipin complex bound to ssG4 with low micromolar affinity and in a selective fashion, as it did not show measurable interactions with a hairpin DNA embedded within the same ssDNA (ssHP), or a mutated ssG4 sequence that had lost the ability to fold into a G4 (ss; Figs 33A and EV2, Appendix Table S2). We next tested a series of G4 sequences found in different genomic contexts and with different folding topologies: we found that the Timeless–Tipin complex bound to all of them, albeit with different affinities that varied several fold, whereas it did not show appreciable binding to ss- or dsDNA (Fig 33B). Figure 3. The Timeless–Tipin complex shows a preference for binding G4 DNAFluorescence anisotropy was used to measure the binding affinity of Timeless–Tipin for the indicated DNA sequences. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA (Appendix Table S2 for sequence details). Binding affinity of Timeless–Tipin for a range of G4 DNA sequences (see Appendix Table S2 for sequence details and references). Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls. Data information: Each data point is the mean of at least 3 independent experiments and the error bars indicate one SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. The Timeless–Tipin complex shows a preference for binding G-quadruplex DNA structures Coomassie-stained SDS–PAGE gel of purified Timeless–Tipin complex. Electrophoretic mobility shift assay (EMSA) showing the binding of Timeless–Tipin to G-quadruplex sequence BU1A + 3.5. Mutation of the G-quadruplex sequence (BU1A + 3.5 mut) disrupts Timeless–Tipin binding (see Appendix Table S2 for sequence details). Timeless–Tipin and DNA are both present at a final concentration of 5 μM. EMSA showing the binding of Timeless–Tipin to G-quadruplex sequences (ssG4, dsG4) but not single-stranded DNA (ss), double-stranded DNA (ds) or hairpin-containing sequences (ssHP, dsHP). Timeless–Tipin and DNA are both present at a final concentration of 5 μM. Download figure Download PowerPoint These findings show that Timeless contains, in its C-terminal half, a previously unrecognised DBD, which closely resembles in structure the tandem repeat of three-helix bundles found in the homeodomain-like superfamily of transcription factors. While Timeless DBD binds to both ss- and dsDNA, it binds with ~ 10-fold greater affinity to a defined G4 DNA sequence. The preference for G4 DNA is retained by the Timeless–Tipin complex. The Timeless C-terminus is crucial for processive G4 replication in vivo To further explore the in vivo contribution of the Timeless C-terminus to G4 replication, we generated a DT40 cell line expressing a version of Timeless truncating the gene before the DBD, using CRISPR/Cas9 gene targeting to exon 16. This truncation also removes a domain previously reported to bind PARP1, the PARP1-binding domain (PBD) (Xie et al, 2015). This cell line exhibited instability of BU-1 expression comparable to the timeless mutant (Fig 44) suggesting a role for the C-terminus of the protein in G4 replication. We confirmed this result by expressing human Timeless truncated at amino acid 816, and thus lacking both the DBD and PBD, in the timeless mutant (Appendix Fig S3). To further dissect this observation, we complemented timeless cells with truncated versions of human Timeless, lacking only the DBD (ΔDBD) or the PBD (PARP*). Timeless lacking either the DBD or the PBD largely restored, although not completely, the BU-1 expression instability of the timeless mutant suggesting that the DBD and PBD act redundantly. Additionally, co-immunoprecipitation (Co-IP) experiments showed that neither region is required for binding the DDX11 helicase (Fig EV3), a known binding partner of Timeless and whose ability to unwind G4s is stimulated by Timeless (Calì et al, 2016). These results indicate that the C-terminus of the Timeless protein has an important role in G4 replication to which both the DBD and the PDB contribute, independently of DDX11 recruitment. Figure 4. The C-terminus of Timeless is required for processive G4 replicationFluctuation analysis for the generation of Bu-1 loss variants. Top to bottom: wild type, timeless (clone 1), a timeless mutant (timeless ∆C) generated by CRISPR-Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP-binding domains. Then, complementation of timeless#1 with human Timeless (hTim), hTim∆816–1,208 (lacking both the DBD and PBD), hTim∆816–965 (lacking the DBD) and hTim[1:1,000], lacking the PBD. At least two independent fluctuation analyses were performed with 24–36 individual clones each cell line per repeat. Bars and whiskers represent median and interquartile range, respectively. *P < 0.05 and ****P < 0.0001; one-way ANOVA for comparison with the wild-type cells. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. The C-terminus of Timeless is not required for its interaction with DDX11HEK293T cells were transiently transfected with a plasmid encoding Flag-hTimeless, or co-transfected with plasmids encoding hDDX11 and Flag-Timeless or with Timeless mutated to delete the DNA-binding domain (ΔDBD: deletion of region S816–S965) or PARP-binding domain (PARP*: truncation at V1000). Twenty-four h after transfection, whole-cell extracts were subjected to immunoprecipitation with anti-Flag magnetic beads. Western Blot analyses were performed to detect overexpressed DDX11 protein in the pulled down samples using a specific antibody. Upper panel: Input and pulled down samples transfected with different Timeless constructs detected with an anti-Flag antibody. Bottom panel: Input and pulled down samples transfected with different Timeless constructs detected with an anti-DDX11 antibody. Tubulin was used as a loading control for the input samples. Download figure Download PowerPoint DDX11 ensures processive G4 replication in vivo Since Timeless itself lacks any catalytic activity, we next explored the extent to which the genetic interaction between Timeless and the DDX11 helicase accounted for G4 processing in this system. As noted above, Timeless interacts with DDX11 (Leman et al, 2010; Calì et al, 2016; Cortone et al, 2018), and this interaction has been shown to be important for sister chromatid cohesion (Cortone et al, 2018) and preservation of fork progression in perturbed condi
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