TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans
2021; Springer Nature; Volume: 40; Issue: 17 Linguagem: Inglês
10.15252/embj.2021108053
ISSN1460-2075
AutoresYisui Xia, Ryo Fujisawa, Tom Deegan, Remi Sonneville, Karim Labib,
Tópico(s)Advanced Data Storage Technologies
ResumoArticle16 July 2021Open Access Transparent process TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans Yisui Xia Yisui Xia orcid.org/0000-0002-5576-2147 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Ryo Fujisawa Ryo Fujisawa orcid.org/0000-0003-1985-1668 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Tom D Deegan Tom D Deegan orcid.org/0000-0001-9639-7636 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Remi Sonneville Corresponding Author Remi Sonneville [email protected] orcid.org/0000-0002-2149-2652 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Karim P M Labib Corresponding Author Karim P M Labib [email protected] orcid.org/0000-0001-8861-379X The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Yisui Xia Yisui Xia orcid.org/0000-0002-5576-2147 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Ryo Fujisawa Ryo Fujisawa orcid.org/0000-0003-1985-1668 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Tom D Deegan Tom D Deegan orcid.org/0000-0001-9639-7636 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Remi Sonneville Corresponding Author Remi Sonneville [email protected] orcid.org/0000-0002-2149-2652 The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Karim P M Labib Corresponding Author Karim P M Labib [email protected] orcid.org/0000-0001-8861-379X The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Author Information Yisui Xia1, Ryo Fujisawa1, Tom D Deegan1, Remi Sonneville *,1 and Karim P M Labib *,1 1The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK **Corresponding author. Tel: +44 1382 386395; E-mail: [email protected] ***Corresponding author. Tel: +44 1382 384108; E-mail: [email protected] The EMBO Journal (2021)40:e108053https://doi.org/10.15252/embj.2021108053 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 The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2LRR-1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 "unfoldase". Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome-associated TIMELESS-TIPIN complex is required for CUL-2LRR-1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS-TIPIN, CUL-2LRR-1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM-7 subunit of CMG. Subsequently, the UBXN-3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4. We show that UBXN-3 is important in vivo for replisome disassembly in the absence of TIMELESS-TIPIN. Correspondingly, co-depletion of UBXN-3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN-3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer. Synopsis Different factors contribute to ubiquitylation-mediated DNA replication termination in different eukaryotic systems. Here, replisome disassembly in Caenorhabditis elegans is found to be stimulated by the replisome-associated TIMELESS-TIPIN complex and the p97/CDC-48 unfoldase adaptor UBXN-3. Caenorhabditis elegans TIM-1_TIPN-1 stimulates CMG helicase ubiquitylation by the CUL-2_LRR-1 ligase in a reconstituted in vitro system. The TIM-1_TIPN-1 complex is important for the association of CUL-2_LRR-1 with the CMG helicase. The TIM-1_TIPN-1 complex stimulates CMG ubiquitylation during replication termination in vivo in the C. elegans early embryo. UBXN-3 stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4 in vitro and in vivo. Introduction Eukaryotic chromosomes are copied just once per cell cycle (Bell & Labib, 2016; Burgers & Kunkel, 2017; Gasser, 2019), dependent upon a dynamic molecular machine known as the replisome (Bai et al, 2017). During S-phase, the replisome assembles around the CMG helicase at nascent DNA replication forks (CMG is named after its three sub-assemblies, namely the CDC-45 protein, the hexameric MCM-2-7 motor that encircles DNA and the GINS complex). After initiation, CMG associates continuously with DNA replication forks throughout elongation (Labib et al, 2000), until termination occurs when two DNA replication forks from neighbouring origins converge, or when a single replisome arrives at a telomere or DNA nick (Maric et al, 2014; Moreno et al, 2014; Dewar et al, 2015; Vrtis et al, 2021). Work with budding yeast and metazoa indicates that the CMG helicase is ubiquitylated on its MCM7 subunit during termination (Maric et al, 2014; Moreno et al, 2014; Dewar et al, 2017; Sonneville et al, 2017). This leads to recruitment of the Cdc48/p97/VCP ATPase via its UFD1-NPL4 adaptor proteins (Franz et al, 2011; Maric et al, 2017; Mukherjee & Labib, 2019; Deegan et al, 2020), which recognise polyubiquitin chains that are linked via lysine 48 of ubiquitin (Bodnar & Rapoport, 2017, Twomey et al, 2019, van den Boom et al, 2016). Cdc48/p97 then unfolds ubiquitylated MCM7 (Deegan et al, 2020), leading to the irreversible dissociation of CMG into its component parts and thus to replisome disassembly and the dissociation of replisome components from DNA. Although metazoa and yeast share common principles of replisome disassembly during DNA replication termination, important differences are also apparent. Disassembly of the budding yeast replisome has been reconstituted with purified proteins, showing that the cullin 1 ligase SCFDia2 directs a single E2 ubiquitin-conjugating enzyme called Cdc34 to initiate and then elongate a long K48-linked ubiquitin chain on CMG-Mcm7 (Maric et al, 2014; Deegan et al, 2020). However, SCFDia2 is absent in metazoa. Work with the nematode Caenorhabditis elegans and the frog Xenopus laevis has shown that a cullin 2 ligase called CUL-2LRR-1 is recruited to the terminating replisome and is required for CMG disassembly during termination (Dewar et al, 2017; Sonneville et al, 2017). The mechanism of CMG ubiquitylation by CUL-2LRR-1 has yet to be determined in any metazoan species and until now the reaction had not been reconstituted in vitro. The very high efficiency of CMG disassembly in budding yeast is enforced by two core replisome components called Ctf4 and Mrc1, which jointly recruit SCFDia2 to the replisome and thereby ensure that every CMG helicase complex is ubiquitylated during termination (Maculins et al, 2015; Deegan et al, 2020). In contrast, a potential role for metazoan core replisome components in stimulating CMG helicase ubiquitylation by CUL-2LRR-1 during DNA replication termination had not previously been explored. Upon ubiquitylation of yeast CMG, the ubiquitin receptors Ufd1-Npl4 recruit Cdc48 and support helicase disassembly, provided that at least five ubiquitin moieties have been conjugated to CMG-Mcm7 (Mukherjee & Labib, 2019; Deegan et al, 2020). Although both yeast Cdc48-Ufd1-Npl4 and human p97-UFD1-NPL4 are sufficient to unfold a model substrate comprising a poly-ubiquitylated fluorescent protein (Blythe et al, 2017; Bodnar & Rapoport, 2017; Pan et al, 2021), work with C. elegans indicated that the disassembly of ubiquitylated CMG helicase by metazoan p97-UFD1-NPL4 is more complicated than would have been predicted by the corresponding studies of their yeast orthologues. A further adaptor of CDC-48/p97 called UBXN-3 was shown to contribute to chromatin unloading of CMG components in worms with reduced expression of CDC-48 (Franz et al, 2016). Moreover, UBXN-3 was found to be required for a second pathway of CMG disassembly that is activated during mitosis (Sonneville et al, 2017). This mitotic CMG disassembly pathway requires the TRUL-1/TRAIP ubiquitin ligase and helps to process sites of incomplete DNA replication (Deng et al, 2019; Priego Moreno et al, 2019; Sonneville et al, 2019). Until now, it was not known whether the role of UBXN-3 in the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4 was direct and the reaction had yet to be reconstituted with purified proteins. In addition, it was unclear whether UBXN-3 also acts during DNA replication termination to stimulate CMG helicase disassembly by CDC-48_UFD-1_NPL-4. Here, we use C. elegans as a model system to explore the mechanism of replisome disassembly during DNA replication termination by metazoan CUL-2LRR-1 and CDC-48_UFD-1_NPL-4. Our data show that the core replisome factors TIMELESS-TIPIN help to recruit CUL-2LRR-1 to the CMG helicase, in order to promote efficient ubiquitylation of CMG-MCM-7. Subsequently, UBXN-3 directly stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4, not only during mitosis but also during DNA replication termination. Lack of both TIMELESS-TIPIN and UBXN-3 causes a synthetic defect in CMG disassembly both in vitro and also in the C. elegans early embryo, with the latter defect being associated with a profound loss of viability. Results An RNAi screen for E2 ubiquitin-conjugating enzymes that work with CUL-2LRR-1 in Caenorhabditis elegans Relatively little is known about the mechanism of C. elegans cullin ligases and the identity of their cognate E2 enzymes. Nevertheless, studies of the equivalent human enzymes have shown that metazoan cullin ligases are considerably more complex than their yeast counterparts and function together with a complex array of different enzymes, in order to synthesise K48-linked ubiquitin chains on their substrates (Baek et al, 2020b; Wang et al, 2020). Firstly, the cullin scaffold must be modified by the ubiquitin-like protein NEDD8, which serves as a nexus that contacts multiple elements of the ligase along with the cognate E2 ubiquitin-conjugating enzyme (Baek et al, 2020a; Wang et al, 2020). Subsequently, specialised "priming" enzymes are responsible for the initial mono-ubiquitylation of substrate lysines, whereas distinct E2 enzymes then mediate the subsequent elongation of K48-linked ubiquitin chains (Kleiger & Deshaies, 2016). Recent work identified two different classes of priming enzymes for human cullin ligases (Fig 1A). The first comprises paralogues of the E2 enzyme UBE2D, which is activated by the RING subunit of a neddylated cullin ligase (Baek et al, 2020a). The second type of priming enzyme is an RBR ("RING-between-RING") E3 ligase of the ARIADNE family, known as ARIH1, which associates with neddylated cullin ligases and receives ubiquitin from the cysteine-specific E2 enzyme UBE2L3, before transferring this ubiquitin to a substrate lysine (Scott et al, 2016; Horn-Ghetko et al, 2021). Subsequently, K48-linked chains are extended on the primed substrate by the human orthologues of yeast Cdc34, known as UBE2R1-2, but these act redundantly with a further E2 enzyme (Fig 1A) called UBE2G1 (Hill et al, 2019). Figure 1. An RNAi screen for candidate E2 enzymes that contribute to CMG-MCM-7 ubiquitylation during DNA replication termination in C. elegans A. Model for the priming and elongation of ubiquitin chains on substrates of cullin ubiquitin ligases in metazoa. See text for details. B. Family tree for the E2 ubiquitin-conjugating enzymes encoded by the C. elegans genome. For each of the indicated groups, a single plasmid was generated to express RNAi to the component genes (see Materials and Methods). C. Summary of RNAi screen to detect synthetic lethality, upon combining ubxn-3 RNAi with a plasmid expressing RNAi to lrr-1 or to one of the groups of E2 enzymes indicated in (B). D. Summary of synthetic lethality data for the screen described in (C). Worms were fed on the indicated proportions of bacteria expressing RNAi to ubxn-3, lrr-1, E2 enzyme groups G1-G6, or else containing empty vector as indicated. The data represent the means and standard deviations from three biological replicates. E, F. Synthetic lethality resulting from the combination of RNAi to E2 group 5 and RNAi to ubxn-3 was deconvolved in similar experiments to those in (D), using plasmids expressing RNAi to individual E2 enzymes, or pairs of E2 enzymes, as indicated. G. GFP-psf-1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments, before preparation of embryonic cell extracts and isolation of GFP-PSF-1 by immunoprecipitation. The indicated factors were monitored by immunoblotting. H. The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disc confocal microscopy (see Materials and Methods), in GFP-psf-1 mCherry-Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ("Control" = empty vector). NEB = nuclear envelope breakdown. The scale bars correspond to 5 µm. I. Analogous experiments to those in (D, E) to deconvolve the synthetic lethality induced by RNAi to ubxn-3 in combination with E2 group 6. Download figure Download PowerPoint The single C. elegans orthologue of UBE2D (LET-70) is essential for worm viability (Zhen et al, 1996), as is the LRR-1 substrate adaptor of CUL-2LRR-1 (Merlet et al, 2010). In contrast, we found that deletion of the sole orthologue of mammalian UBE2R1/R2 in C. elegans was viable (ubc-3, Appendix Fig S1A–B and G–H), as was deletion of worm UBE2G1 (ubc-7, Appendix Fig S1C–D and G–H), or mutation of worm UBE2L3 (ubc-18) at a site predicted to abrogate its interaction with E1 (Fay et al, 2003). To investigate which of the C. elegans E2 enzymes might function in vivo with CUL-2LRR-1 during DNA replication termination, we developed a genetic screen that was based on our previous observation that partial RNAi inactivation of lrr-1 is synthetic lethal with RNAi depletion of UBXN-3 (Sonneville et al, 2017). This indicated that RNAi depletion of E2 enzymes that act with CUL-2LRR-1 during DNA replication termination might also cause synthetic lethality in combination with ubxn-3 RNAi. We divided the worm E2 enzymes into a series of phylogenetic groups and constructed RNAi plasmids to inactivate each group simultaneously (Fig 1B). We then fed worms on bacteria expressing ubxn-3 RNAi or empty vector (Fig 1C), mixed one-to-one either with bacteria expressing each of the six groups of E2 RNAi, or with positive and negative controls (10% lrr-1 RNAi and empty vector, respectively). Most of the tested E2 groups had no impact on viability together with ubxn-3 RNAi, whereas the combination of lrr-1 and ubxn-3 RNAi reduced viability close to zero (Fig 1D). However, E2 group 5 produced a strong synthetic lethal phenotype in combination with ubxn-3 (Fig 1D), which subsequent deconvolution showed was dependent upon the combined inactivation of ubc-3 and ubc-7 (Fig 1E and F). Triple RNAi inactivation of ubxn-3, ubc-3 and ubc-7 led to the partial accumulation of the CMG helicase with short ubiquitin chains on MCM-7 (Fig 1G), and the partial retention of CMG on chromatin during mitosis (Fig 1H). Moreover, co-depletion of UBC-3 and UBC-7 reduced the ubiquitylation of CMG-MCM-7 in worms that had been treated with npl-4 RNAi in order to block CMG disassembly by CDC-48 (Appendix Fig S2). These findings indicated that UBC-3 and UBC-7 contribute to CMG ubiquitylation and disassembly in the C. elegans early embryo, acting redundantly with each other. Whereas deletion of cul-2 or lrr-1 is lethal in C. elegans (Feng et al, 1999; Merlet et al, 2010), the combination of ubc-3∆ and ubc-7∆ is viable although the brood size is reduced (Appendix Fig S1G and H). This indicates that other E2 enzymes must be able to act with CUL-2LRR-1, in addition to UBC-3 and UBC-7. One candidate for the latter is the essential LET-70 orthologue of mammalian UBE2D enzymes. A further possibility was suggested by the ˜50% synthetic lethality produced by RNAi to ubxn-3 plus E2 group 6 (Fig 1D), which subsequent deconvolution showed was due to the ubc-18 orthologue of human UBE2L3 (Fig 1I). This suggested a role for an RBR ligase of the ARIADNE family. Reconstitution of replisome-dependent CMG ubiquitylation by CUL-2LRR-1 and a suite of ubiquitylation and neddylation enzymes In order to explore the mechanism by which CUL-2LRR-1 promotes replisome-dependent ubiquitylation of the C. elegans CMG helicase, we set out to reconstitute the reaction with purified proteins, taking advantage of recent findings in addition to the results of the E2 screen described above. Work with budding yeast (Deegan et al, 2020) and Xenopus laevis (Low et al, 2020) has shown that CMG ubiquitylation is stimulated by release of the helicase from replication fork DNA, likely reflecting the events that normally lead to CMG disassembly and the termination of DNA replication, when a fork reaches a DNA end such as a telomere or a nick in the DNA template strand upon which the helicase tracks. Based on this observation, replisome-dependent ubiquitylation of the yeast CMG helicase was recently reconstituted with purified proteins in the complete absence of DNA (Deegan et al, 2020), reflecting the inherently high efficiency of CMG ubiquitylation, which is repressed throughout elongation by the embrace of the helicase with a replication fork. Encouraged by these studies, we expressed and purified recombinant forms of the C. elegans CMG helicase and associated core replisome proteins (Fig 2A). We also purified a range of ubiquitylation enzymes, together with C. elegans NED-8 (equivalent to mammalian NEDD8) and the worm orthologues of mammalian neddylation enzymes (Fig 2A, ULA-1_RFL-1, UBC-12 and DCN-1). Both ubiquitylation and neddylation require an E1 enzyme to activate ubiquitin/NEDD8, which is then transferred to the catalytic cysteine residue of an E2 enzyme (Rennie et al, 2020). Subsequently, E3 enzymes mediate the transfer of ubiquitin or NEDD8 from activated E2 to substrate lysine residues (Morreale & Walden, 2016; Zheng & Shabek, 2017), either by bringing E2 and substrate into close proximity and stabilising the active conformation of the E2-Ub or E2-NEDD8 conjugate (e.g. RING E3 ligases including the cullin family), or by transfer of ubiquitin onto a cysteine residue of the E3 and thereafter onto a proximal substrate lysine (e.g. HECT or RBR E3 ligases). Figure 2. In vitro reconstitution of replisome-specific ubiquitylation of CMG-MCM-7 by CUL-2LRR-1 and an array of associated ubiquitylation enzymes Purified C. elegans proteins (see Materials and Methods). Ubiquitylation of CMG-MCM-7 was reconstituted with the indicated factors as described in Materials and Methods. "Neddylation" indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. Equivalent reactions to those in (B) but using lysine-free (K0) ubiquitin. Similar reactions comparing wild-type ubiquitin to the indicated ubiquitin mutants. Reactions containing CMG and either CUL-2LRR-1 or CUL-2VHL-1 were performed in the presence or absence of other replisome factors as indicated. CMG was then isolated by immunoprecipitation of SLD-5 and the indicated factors were monitored by immunoblotting. Download figure Download PowerPoint Guided by the analysis of C. elegans E2 enzymes described above, we compared the ability of UBC-3, UBC-18 and LET-70 to support the ubiquitylation of recombinant C. elegans CMG in reconstituted in vitro reactions, which also contained E1, CUL-2LRR-1, the worm neddylation machinery and other replisome factors (Fig 2A). In reactions containing UBC-3 as the only E2 enzyme, ubiquitylation of CMG-MCM-7 was not observed (Fig 2B, compare lanes 1 and 5). In contrast, UBC-18 together with the RBR ligase ARI-1 supported the addition of 1–3 ubiquitins to MCM-7 (Fig 2B lane 2; Fig EV1B lanes 9–12 show that both UBC-18 and ARI-1 were required for MCM-7 ubiquitylation). This represented mono-ubiquitylation of multiple sites on MCM-7, since the same ubiquitylation pattern was observed with lysine-free ubiquitin (Fig EV1A, compare lanes 2–3). LET-70 also supported CMG ubiquitylation, with up to ˜8 ubiquitins being conjugated to MCM-7 (Fig 2B, lane 3). This predominantly represented the conjugation by LET-70 of a single ubiquitin chain on CMG-MCM-7, since mono-ubiquitylation was the major product in reactions containing LET-70 and lysine-free ubiquitin (Fig EV1A, compare lanes 6–7). Moreover, the chains formed by LET-70 were largely independent of lysine 48 of ubiquitin (Fig EV1A, compare lanes 6 and 8). These findings indicated that UBC-18_ARI-1 and LET-70 are both able to prime ubiquitylation of CMG-MCM-7, but cannot synthesise the K48-linked ubiquitin chains that are the preferred substrate of p97-UFD1-NPL4 (Bodnar & Rapoport, 2017, Tsuchiya et al, 2017, van den Boom et al, 2016). Click here to expand this figure. Figure EV1. The role of C. elegans LET-70, ARI-1_UBC-18 and cullin neddylation in reconstituted CMG-MCM-7 ubiquitylation A, B. Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as in Fig 2, in the presence of the indicated factors. "Neddylation" indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. C. Similar reactions were performed in the presence of the indicated E2 enzymes and ubiquitin variants. D. Comparison of purified CUL-2LRR-1 containing either wt CUL-2 or CUL-2-2R. The latter has mutation of lysine 719 and lysine K479, which in human CUL-2 comprise a neddylation site and a site of interaction with the DCN-1 E3 ligase for neddylation (Bandau et al, 2012). We previously showed that the combined mutation of these two sites prevents human CUL-2 from supporting CMG-MCM-7 ubiquitylation in Xenopus egg extracts (Sonneville et al, 2017). E. The ability of CUL-2LRR-1, CUL-2-2RLRR-1 and CUL-2VHL-1 to support the formation of free ubiquitin chains by UBC-3 was monitored in reactions containing FLAG-tagged ubiquitin and the indicated factors. CUL-2 neddylation and the formation of ubiquitin chains was then monitored by immunoblotting with anti-CUL-2 or anti-FLAG antibodies. F. CMG-MCM-7 ubiquitylation reactions were performed as above with the indicated factors. Data information: Wt = wild-type ubiquitin; K0 = all lysines mutated to arginine; K48R = lysine 48 mutated to arginine; K48-only = all lysines of ubiquitin mutated to arginine except for lysine 48. Download figure Download PowerPoint We then performed similar reactions containing CUL-2LRR-1 with combinations of UBC-3, LET-70 and UBC-18_ARI-1. These experiments showed that UBC-3 is able to synthesise long ubiquitin chains on CMG-MCM-7, dependent upon the chains having been initiated by either LET-70 or UBC-18_ARI-1 (Fig 2B, lanes 6–7). MCM-7 ubiquitylation in the presence of UBC-3 predominantly takes the form of K48-linked ubiquitin chains (Fig 2D). Moreover, the major product in reactions containing all three E2 enzymes, together with the E3 ligases CUL-2LRR-1 and ARI-1, is a single polyubiquitin chain on MCM-7, since reactions containing lysine-free ubiquitin only supported the conjugation of a single ubiquitin moiety to most MCM-7 molecules (compare Fig 2B lane 8 with Fig 2C lane 8). Therefore, these data indicate that LET-70 is the predominant priming enzyme under these reaction conditions (Fig 2C, compare lane 3 with lanes 6–8), whereas UBC-3 is the predominant E2 that extends K48-linked ubiquitin chains on CMG-MCM-7. Consistent with the in vivo results of the RNAi E2 screen, we observed in similar in vitro reactions that UBC-7 was also able to elongate K48-linked ubiquitin chains that had been primed by LET-70, although UBC-7 was less efficient than UBC-3 (Fig EV1C). In contrast, the more distantly related E2 enzymes UBC-1 and UBC-14 were unable to promote CMG-MCM-7 ubiquitylation (Fig EV1C), as predicted by the RNAi E2 screen (Fig 1C–E). CMG-MCM-7 ubiquitylation by both UBC-18_ARI-1 and UBC-3 was stimulated by neddylation of conserved lysine residues on CUL-2 (Fig EV1-EV5D–F), consistent with previous studies of human CUL-2 (Bandau et al, 2012; Sonneville et al, 2017). This contrasts with the action of yeast SCFDia2 for which neddylation is dispensable (Mukherjee & Labib, 2019; Deegan et al, 2020). Interestingly, CMG-MCM-7 ubiquitylation by C. elegans LET-70 did not require neddylation (Fig EV1B, compare lanes 1–4). We found that this was because LET-70 is able to ubiquitylate the neddylation sites on CUL-2 (Fig EV1B lanes 1–2), indicating that cullin ubiquitylation can functionally substitute for cullin neddylation. Correspondingly, mutation of K719 and K749 of CUL-2 abrogated the ubiquitylation or neddylation of CUL-2 and also blocked CMG ubiquitylation (Fig EV1-EV5D–F; Fig EV1E shows that CUL-2-2RLRR-1 and wild-type CUL-2LRR-1 are equally active at promoting free chain formation by UBC-3 in the absence of neddylation). Click here to expand this figure. Figure EV2. C. elegans TIM-1_TIPN-1 promotes the priming of CMG-MCM-7 ubiquitylation by LET-70 and ARI-1_UBC-18 Purified CMG, TIM-1_TIPN-1, POLɛ, CLSP-1, CTF-18_RFC, CTF-4, MCM-10 and CUL-2LRR-1 were mixed before immunoprecipitation of CMG using polyclonal antibodies to SLD-5. The association of CMG with other replisome factors was then monitored by immunoblotting. Three repeats of the experiment in Fig 3B. Quantification of the data in (B). For each sample, the proportion of modified MCM-7 was calculated, relative to unmodified MCM-7. The panel shows the mean values from the three repeats, together with the standard deviation. Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as in Fig 3, in the presence of the indicated factors (including 15 nM CUL-2LRR-1). "Neddylation" indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. Similar reactions were performed in the presence of 3 nM CUL-2LRR-1, to reveal the contribution of TIM-1_TIPN-1 to priming of CMG-MCM-7 ubiquitylation by LET-70. Glycerol gradient analysis of the indicated protein mixtures, in the presence of annealed oligonucleotides with 45 bp double-strand DNA and 39nt single-strand 3' flap DNA (see Materials and Methods and Appendix Table S1). In reactions analogous to those in Fig 3E and F, ubiquitylation of the indicated factors was monitored by immunoblotting. Reactions were performed in sets of three as indicated (1 = dropout of CUL-2LRR-1; 2 = wt ubiquitin; 3 = lysine-free or K0 ubiquitin). Ubiquitylation of the TIM-1_TIPN-1 complex was monitored in the presence or absence of the indicated factors. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. C. elegans CMG disassembly is dependent upon polyubiquitylation and the UBX domain of UBXN-3 Reactions were performed as described for Fig 4, in the presence of the indicated factors. "Neddylation" indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. The bands corresponding to MCM-7 conjugated to 1–5 ubiquitins are indicated in lane 6. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Deletion of the C. elegans ctf-4 gene and RNAi depletion of CSLP-1 does not impair the in vivo ubiquitylation of CMG-MCM-7 during DNA replication termination Illustration of the region of the C. elegans ctf-4 gene that was deleted by CRISPR-Cas9. Arrows indicate PCR primers that were used to monitor the deletion in adult worms. PCR analysis of wt and ctf-4∆ worms with the indicated primers (non-specific bands are denoted with asterisks). Control or ctf-4∆ worms were subjected to RNAi to npl-4 and clsp-1 as indicated, before isolation of TAP-PSF-1 from cell extracts. The association of the indicated factors with TAP-PSF-1 was monitored by immunoblotting, together with ubiquitylation of CMG-MCM-7. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. RNAi inactivation of tim-1 in trul-1∆ worms does not lead to persistence of CMG on mitotic chromatin The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disc confocal microscopy (see Materials and Methods), in trul-1∆ worms exposed to the indicated RNAi treatments ("Control" = worms fed on bacteria containing empty vector). The scale bars correspond to 5 µm. Quantification of the data in (A). Embryonic viability was measured as above for the indicated RNAi treatments of wild-type (control) or trul-1∆ worms. The data represent the means and standard deviations from three biologi
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