CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis

Abstract

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.

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Figure 1: The CDC-48 cofactor NPL-4 is required for CMG helicase disassembly during S phase in the C. elegans early embryo.
Figure 2: CUL-2LRR-1 is required for CMG helicase disassembly during S phase in C. elegans.
Figure 3: A mitotic pathway for CMG helicase disassembly is revealed in the absence of CUL-2LRR-1.
Figure 4: The mitotic CMG helicase disassembly pathway requires UBXN-3 and is modulated by the SUMO protease ULP-4, both of which become essential when LRR-1 is depleted.
Figure 5: Isolation of the post-termination worm replisome.
Figure 6: CUL2LRR1 associates with the post-termination vertebrate replisome and is recruited to chromatin during DNA replication termination in Xenopus egg extracts.
Figure 7: Active CUL2LRR1 is required for extraction of CMG components from chromatin during DNA replication termination in Xenopus egg extracts.

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Acknowledgements

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.

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Contributions

R.S. performed the experiments in Figs 15 and Supplementary Figs 14. S.P.M. performed the experiments in Figs 6 and 7 and Supplementary Fig. 5. K.L. and A.Gambus conceived the project and designed experiments in collaboration with R.S. and S.P.M. A.K. and C.J. produced recombinant CUL2–RBX1 and C.J.H. provided recombinant LRR1. A.Gartner provided invaluable support in the early stages of the project. K.L. wrote the manuscript, with contributions and critical comments from the other authors.

Corresponding authors

Correspondence to Agnieszka Gambus or Karim Labib.

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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.

Supplementary Figure 3 A new pathway for CMG helicase disassembly acts during mitosis.

(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).

Supplementary Figure 5 Additional supplementary material for experiments with Xenopus egg extracts.

(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.

Supplementary Figure 6 CUL2 is very highly conserved in vertebrates.

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.

(ad) 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.

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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)

FRAP analysis of GFP-CDC-45 after depletion of NPL-4.

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)

GFP-PSF-1 remains on chromatin throughout mitosis in embryos depleted for both UBXN-3 and LRR-1.

Images were acquired and analysed as for Supplementary Movie 1. (MOV 3887 kb)

GFP-PSF-1 is released from chromatin before prophase in ubxn-3 RNAi embryos.

Images were acquired and analysed as for Supplementary Movie 1. (MOV 3219 kb)

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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

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