Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (CDC45–MCM–GINS). Using Caenorhabditis elegans early embryos and Xenopus laevis egg extracts, we show that the E3 ligase CUL-2LRR-1 associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2LRR1 as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2LRR-1, but is then removed by a mitotic pathway that requires the CDC-48 cofactor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gambus, A. et al. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat. Cell Biol. 8, 358–366 (2006).
Moyer, S. E., Lewis, P. W. & Botchan, M. R. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl Acad. Sci. USA 103, 10236–10241 (2006).
Bell, S. P. & Labib, K. Chromosome duplication in Saccharomyces cerevisiae. Genetics 203, 1027–1067 (2016).
Deegan, T. D. & Diffley, J. F. MCM: one ring to rule them all. Curr. Opin. Struct. Biol. 37, 145–151 (2016).
O’Donnell, M. & Li, H. The eukaryotic replisome goes under the microscope. Curr. Biol. 26, R247–R256 (2016).
Labib, K., Tercero, J. A. & Diffley, J. F. X. Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000).
Ilves, I., Petojevic, T., Pesavento, J. J. & Botchan, M. R. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol. Cell 37, 247–258 (2010).
Bell, S. P. DNA replication. Terminating the replisome. Science 346, 418–419 (2014).
Dewar, J. M., Budzowska, M. & Walter, J. C. The mechanism of DNA replication termination in vertebrates. Nature 525, 345–350 (2015).
Maric, M., Maculins, T., De Piccoli, G. & Labib, K. Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication. Science 346, 1253596 (2014).
Moreno, S. P., Bailey, R., Campion, N., Herron, S. & Gambus, A. Polyubiquitylation drives replisome disassembly at the termination of DNA replication. Science 346, 477–481 (2014).
Maculins, T., Nkosi, P. J., Nishikawa, H. & Labib, K. Tethering of SCF(Dia2) to the replisome promotes efficient ubiquitylation and disassembly of the CMG helicase. Curr. Biol. 25, 2254–2259 (2015).
Morohashi, H., Maculins, T. & Labib, K. The amino-terminal TPR domain of Dia2 tethers SCF(Dia2) to the replisome progression complex. Curr. Biol. 19, 1943–1949 (2009).
Soucy, T. A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736 (2009).
Duda, D. M. et al. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008).
Saha, A. & Deshaies, R. J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008).
Sonneville, R., Querenet, M., Craig, A., Gartner, A. & Blow, J. J. The dynamics of replication licensing in live Caenorhabditis elegans embryos. J. Cell Biol. 196, 233–246 (2012).
Sonneville, R., Craig, G., Labib, K., Gartner, A. & Blow, J. J. Both chromosome decondensation and condensation are dependent on DNA replication in C. elegans embryos. Cell Rep. 12, 405–417 (2015).
Avci, D. & Lemberg, M. K. Clipping or extracting: two ways to membrane protein degradation. Trends Cell Biol. 25, 611–622 (2015).
Franz, A., Ackermann, L. & Hoppe, T. Ring of change: CDC48/p97 drives protein dynamics at chromatin. Front. Genet. 7, 73 (2016).
Ramadan, K., Halder, S., Wiseman, K. & Vaz, B. Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication. Chromosoma http://dx.doi.org/10.1007/s00412-016-0587-4 (2016).
Meyer, H. H., Shorter, J. G., Seemann, J., Pappin, D. & Warren, G. A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, 2181–2192 (2000).
Mouysset, J., Kahler, C. & Hoppe, T. A conserved role of Caenorhabditis elegans CDC-48 in ER-associated protein degradation. J. Struct. Biol. 156, 41–49 (2006).
Sarikas, A., Hartmann, T. & Pan, Z. Q. The cullin protein family. Genome Biol. 12, 220 (2011).
Kloppsteck, P., Ewens, C. A., Forster, A., Zhang, X. & Freemont, P. S. Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family. Biochim. Biophys. Acta 1823, 125–129 (2012).
Meyer, H., Bug, M. & Bremer, S. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 14, 117–123 (2012).
Vaz, B., Halder, S. & Ramadan, K. Role of p97/VCP (Cdc48) in genome stability. Front. Genet. 4, 60 (2013).
Pelisch, F. et al. Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans. Nat. Commun. 5, 5485 (2014).
Merlet, J. et al. The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development. Development 137, 3857–3866 (2010).
Starostina, N. G., Simpliciano, J. M., McGuirk, M. A. & Kipreos, E. T. CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells. Dev. Cell 19, 753–764 (2010).
Fullbright, G., Rycenga, H. B., Gruber, J. D. & Long, D. T. p97 promotes a conserved mechanism of helicase unloading during DNA cross-link repair. Mol. Cell. Biol. 36, 2983–2994 (2016).
Semlow, D. R., Zhang, J., Budzowska, M., Drohat, A. C. & Walter, J. C. Replication-dependent unhooking of DNA interstrand cross-links by the NEIL3 glycosylase. Cell 167, 498–511 (2016).
Cuvier, O., Stanojcic, S., Lemaitre, J. M. & Mechali, M. A topoisomerase II-dependent mechanism for resetting replicons at the S-M-phase transition. Genes Dev. 22, 860–865 (2008).
Bandau, S., Knebel, A., Gage, Z. O., Wood, N. T. & Alexandru, G. UBXN7 docks on neddylated cullin complexes using its UIM motif and causes HIF1α accumulation. BMC Biol. 10, 36 (2012).
Ossareh-Nazari, B., Katsiarimpa, A., Merlet, J. & Pintard, L. RNAi-based suppressor screens reveal genetic interactions between the CRL2LRR-1 E3-ligase and the DNA replication machinery in Caenorhabditis elegans. G3 (Bethesda) 6, 3431–3442 (2016).
Dewar, J. M., Low, E., Mann, M., Raschle, M. & Walter, J. C. CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination. Genes Dev. http://dx.doi.org/10.1101/gad.291799.116 (2017).
Franz, A. et al. CDC-48/p97 coordinates CDT-1 degradation with GINS chromatin dissociation to ensure faithful DNA replication. Mol. Cell 44, 85–96 (2011).
Franz, A. et al. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression. Nat. Commun. 7, 10612 (2016).
Lee, J. J. et al. Complex of Fas-associated factor 1 (FAF1) with valosin-containing protein (VCP)-Npl4-Ufd1 and polyubiquitinated proteins promotes endoplasmic reticulum-associated degradation (ERAD). J. Biol. Chem. 288, 6998–7011 (2013).
Menges, C. W., Altomare, D. A. & Testa, J. R. FAS-associated factor 1 (FAF1): diverse functions and implications for oncogenesis. Cell Cycle 8, 2528–2534 (2009).
Sharrock, W. J., Sutherlin, M. E., Leske, K., Cheng, T. K. & Kim, T. Y. Two distinct yolk lipoprotein complexes from Caenorhabditis elegans. J. Biol. Chem. 265, 14422–14431 (1990).
Gambus, A. et al. A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome. EMBO J. 28, 2992–3004 (2009).
Sengupta, S., van Deursen, F., de Piccoli, G. & Labib, K. Dpb2 integrates the leading-strand DNA polymerase into the eukaryotic replisome. Curr. Biol. 23, 543–552 (2013).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).
Edgley, M. L., Baillie, D. L., Riddle, D. L. & Rose, A. M. Genetic balancers. WormBook 6, 1–32 (2006).
Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).
Gillespie, P. J., Gambus, A. & Blow, J. J. Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins. Methods 57, 203–213 (2012).
Gambus, A., Khoudoli, G. A., Jones, R. C. & Blow, J. J. MCM2-7 form double hexamers at licensed origins in Xenopus egg extract. J. Biol. Chem. 286, 11855–11864 (2011).
Khoudoli, G. A. et al. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition. Curr. Biol. 18, 838–843 (2008).
Prokhorova, T. A. & Blow, J. J. Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity. J. Biol. Chem. 275, 2491–2498 (2000).
Heubes, S. & Stemmann, O. The AAA-ATPase p97-Ufd1-Npl4 is required for ERAD but not for spindle disassembly in Xenopus egg extracts. J. Cell Sci. 120, 1325–1329 (2007).
Walter, J. C. Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts. J. Biol. Chem. 275, 39773–39778 (2000).
Hodgson, B., Li, A., Tada, S. & Blow, J. J. Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import. Curr. Biol. 12, 678–683 (2002).
Lee, D. W. et al. The Dac-tag, an affinity tag based on penicillin-binding protein 5. Anal. Biochem. 428, 64–72 (2012).
We gratefully acknowledge the support of the Medical Research Council (core grant MC_UU_12016/13 for K.L.; award MR/K007106/1 to A.Gambus) the Wellcome Trust (reference 102943/Z/13/Z for award to K.L.; reference 0909444/Z/09/Z for award to A.Gartner) and the Lister Institute (award to A.Gambus) for funding our work. We thank J. Blow for geminin protein, MRC PPU reagents (https://mrcppureagents.dundee.ac.uk) for recombinant frog LRR1 and for producing antibodies, and T. Deegan for helpful comments on the manuscript. We also thank L. Pintard (Institut Jacques Monod, France) for providing the worm line heterozygous for lrr-1Δ, C. Ponting for advice regarding orthologues of the budding yeast Dia2 protein, and J. Walter and E. Low for discussing unpublished findings.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 The CDC-48_UFD-1_NPL-4 complex is required for CMG helicase disassembly in C. elegans.
(a) cdc-48 RNAi leads to persistence of GINS and CDC-45 on chromatin during prophase and throughout mitosis (examples indicated by arrows). (b) Adaptors of CDC-48 in C. elegans. (c) ufd-1 RNAi leads to persistence of GINS and CDC-45 on chromatin during prophase and throughout mitosis (examples indicated by arrows). (d) Equivalent experiment to that in Figure 1d, illustrating the effect of npl-4 RNAi on embryos expressing GFP-MCM-3. To help visualise the small proportion of GFP-MCM-3 on chromatin in early metaphase (marked by an arrow), the experiment also included RNAi to the 3’UTR of endogenous MCM3 (this 3’ UTR is not present in the GFP-MCM-3 transgene), to increase the incorporation of GFP-MCM3 into replication complexes. (e) cdc-48 RNAi experiment, analogous to that in Figure 1d. (f) Homozygous GFP-psf-1 worms were exposed to the indicated RNAi. Embryos were then isolated and processed as in Figure 1e-f. The middle panels show that the amount of CMG isolated from RNR-1 depleted extract was reduced compared to control (compare levels of MCM-7, MCM-2 and CDC-45), due to the inhibition of DNA replication in each embryonic cell cycle. In the right panels, loading of the GFP-PSF-1 IP samples was adjusted to obtain a similar level of CMG (compare MCM-2 and CDC-45). (g,h) Photobleaching experiments for GFP-SLD5 and GFP-MCM3, equivalent to the experiment in Figure 1h. The scale bars correspond to 5 μm. Unprocessed scans of key immunoblots are shown in Supplementary Figure 8.
Supplementary Figure 2 CUL-2LRR-1 is required for removal of GINS from chromatin during S-phase in C. elegans.
(a) C. elegans contain six families of cullin complexes, each with a specific cullin and a unique set of substrate adaptors. (b) Embryos from GFP-sld-5 mCherry-H2B worms were exposed to RNAi against the indicated cullins and processed as in Figure 2. Timelapse images are shown from S-phase to mid-prophase. (c) Six forms of the CUL-2 ligase in C. elegans, each with a unique substrate adaptor. (d) Embryos from GFP-sld-5 mCherry-H2B worms were exposed to RNAi against the indicated substrate adaptors of CUL-2 and processed as in Figure 2. Timelapse images are shown from S-phase to mid-prophase. RNAi for zyg-11 produces meiotic defects and leads to abnormal nuclear morphology in the first embryonic cell cycle. Arrows in this figure indicate the persistent association of GFP-SLD-5 with mitotic chromatin in embryos treated with npl-4 RNAi. Scale bars correspond to 5 μm.
(a) Embryos from GFP-psf-1 mCherry-H2B worms were exposed to the indicated RNAi and processed as in Figure 3. Timelapse images of the first embryonic cell cycle are shown from S-phase to metaphase. GFP-PSF1 initially persists on prophase chromatin following RNAi to components of CUL-2LRR-1 (the arrows denote examples), before being released in late prophase (indicated by asterisks). (b) Extended timecourses for the GFP-SLD-5 data presented in Figure 2a, b. (c) Data from the first cell cycle, for the experiment in Figure 3b. (d) Embryos from GFP-cdc-45 mCherry-H2B worms were exposed to the indicated RNAi and processed as above. (e) Illustration of CMG disassembly defects produced either by depletion of CDC-48/UFD-1/NPL-4, or by depletion of components of CUL-2LRR-1. Scale bars correspond to 5 μm.
Supplementary Figure 4 The mitotic disassembly pathway for the CMG helicase requires UBXN-3 and is modulated by ULP-4.
(a) Embryos from GFP-sld-5 mCherry-H2B worms were exposed to the indicated RNAi and processed as in Figure 4a. The arrows indicate persistent association of GFP-PSF1 with mitotic chromatin (throughout mitosis in the case of RNAi to npl-4, or after simultaneous RNAi to lrr-1 + ubxn-3), whereas the asterisk denotes release of GFP-PSF-1 from chromatin in late prophase in embryos treated only with lrr-1 RNAi. Scale bars correspond to 5 μm. (b) Embryos from GFP-cdc-45 mCherry-H2B worms were processed as for Figure 4b. (c) Embryos from GFP-psf-1 mCherry-H2B worms were exposed to the indicated RNAi and processed as above. The data correspond to the AB cell in the second cell cycle and CMG components remained on chromatin until at or after nuclear envelope breakdown in 3/5 embryos treated with lrr-1 ulp-4 double RNAi. The panel shows an example of an embryo where CMG persists on chromatin until nuclear envelope breakdown upon co-depletion of LRR-1 and ULP-4. (d) Data from a similar experiment, corresponding to the EMS cell in the third cell cycle. Note that in this case we also depleted the ATL-1 checkpoint kinase, to shorten the otherwise long cell cycle delay that is induced by the combination of ulp-4 lrr-1 double RNAi. CMG components remained on chromatin until late metaphase in 5/5 embryos treated with lrr-1 ulp-4 atl-1 triple RNAi. CMG was extracted normally from chromatin during S-phase in embryos subjected to ulp-4 atl-1 double RNAi (5/5 embryos tested), whereas lrr-1 atl-1 double RNAi resembled lrr-1 single RNAi treatment (CMG extracted before the end of prophase in 5/5 embryos).
(a) In a similar experiment to that in Figure 6c, replisome disassembly was blocked during chromosome replication by addition of MLN4924 to Xenopus egg extracts. After isolation of chromatin and digestion of DNA, immunoprecipitation of LRR1 led to co-depletion of CUL2. (b) Analysis of ongoing DNA synthesis at the indicated timepoints for the experiment in Figure 6e, f, by addition of short pulses of α-dATP (see Methods). Data for repeats of this experiment are included in Supplementary Table 6. (c) Replication kinetics for the experiment in Figure 6h, measured by monitoring total incorporation of α-dATP into nascent DNA by the indicated timepoints (see Methods). Data for repeats of this experiment are included in Supplementary Table 6. Unprocessed scans of key immunoblots from this Figure are shown in Supplementary Figure 8.
Alignment of Xenopus CUL2 with the human and mouse orthologues, showing that the mammalian and frog proteins are almost identical. Moreover, previous work indicated that all key residues in CUL2 that contact EloB-C and substrate adaptors are 100% conserved between the human and frog orthologues1.
Supplementary Figure 7 Validation of new antibodies generated in this study for C. elegans proteins.
(a–d) In each case, RNAi was used to deplete the corresponding protein, before immunoblotting of embryonic extracts (upper panels). Ponceau S staining of the nitrocellulose membare (lower panels) provides a loading control in each case.
Supplementary Information (PDF 5929 kb)
Supplementary Information (XLSX 34 kb)
Supplementary Information (XLSX 33 kb)
Supplementary Information (XLSX 34 kb)
Supplementary Information (XLSX 33 kb)
Supplementary Information (XLSX 33 kb)
Supplementary Information (XLSX 13 kb)
Supplementary Information (XLSX 33 kb)
Supplementary Information (XLSX 43 kb)
The CMG helicase component PSF-1 does not associate with condensing chromatin during mitotic prophase or throughout mitosis.
Video of a single optical section through an embryo expressing GFP-PSF-1 (left panel) and mCherry-Histone H2B (right panel) progressing throughout the first and second embryonic cell cycles. Images were acquired every 10 sec with a spinning disk confocal microscope and processed with ImageJ software. The female and male pronuclei are orientated respectively towards the left and right of the video. (MOV 2692 kb)
GFP-PSF-1 associates with condensing chromatin during prophase in embryos depleted for NPL-4 and remains on chromatin throughout mitosis.
Images were acquired and analysed as for Supplementary Movie 1. (MOV 5257 kb)
The movie was generated as above and shows an embryo expressing GFP-CDC-45 (left panel) and mCherry-Histone H2B (right panel). The female pronucleus (left side of the embryo) was photobleached during early S-phase (shown as a white disk in the video at 1’50”) and the chromosomes from the female and male pronuclei were then analysed during the following mitosis (see 19’30” to 24’50”). No recovery of the GFP-CDC-45 signal was observed on the female chromatin, indicating that depletion of NPL-4 causes CDC-45 to persist on chromatin from S-phase until the end of mitosis. (MOV 1176 kb)
GFP-PSF-1 associates with condensing chromatin during prophase in embryos depleted for CUL-2, but is then released from chromatin during late prophase.
Images were acquired and analysed as for Supplementary Movie 1. Note that depletion of CUL-2 leads to meiotic defects in the embryo and thus to abnormal nuclear morphology, reflecting the important role of CUL-2ZYG-11 during the second meiotic cell division2,3. In addition, mitotic entry is delayed in the first embryonic cell cycle after depletion of CUL-2. (MOV 3596 kb)
GFP-PSF-1 associates with condensing chromatin during prophase in embryos depleted for LRR-1, but is then released from chromatin during late prophase.
Images were acquired and analysed as for Supplementary Movie 1. The association of GFP-PSF-1 with prophase chromatin can be seen in the first embryonic cell cycle (P0 cell) from 3’20” to 5’50” and during the second cell cycle from 24’30” to 26’10” for the AB cell (left side of embryo) or from 27’30” to 29’10” for the P1 cell (right side). (MOV 2271 kb)
Images were acquired and analysed as for Supplementary Movie 1. (MOV 3887 kb)
Images were acquired and analysed as for Supplementary Movie 1. (MOV 3219 kb)
About this article
Cite this article
Sonneville, R., Moreno, S., Knebel, A. et al. CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis. Nat Cell Biol 19, 468–479 (2017). https://doi.org/10.1038/ncb3500
Biochemical Society Transactions (2020)
Trends in Genetics (2020)
CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold
EMBO reports (2020)