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Cited by 227DNA Repair MechanismsUbiquitin and proteasome pathwaysEpigenetics and DNA Methylation

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Replication fork slowing and reversal upon DNA damage require PCNA polyubiquitination and ZRANB3 DNA translocase activity.

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Vujanovic, Marko, Krietsch, Jana, Raso, Maria Chiara, Terraneo, Nastassja, Zellweger, Ralph, Schmid, Jonas A., Taglialatela, Angelo, Huang, Jen-Wei, Holland, Cory L., Zwicky, Katharina, Herrador, Raquel, Jacobs, Heinz, Cortez, David, Ciccia, Alberto, Penengo, Lorenza, Lopes, Massimo·2017·Molecular Cell
DOI 10.1016/j.molcel.2017.08.010PMC PMC5594246
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Abstract

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

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Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 PCNA Ubiquitination Is Required for Replication Fork Slowing and Reversal upon Genotoxic Stress (A) Labeling scheme of DNA fiber experiments: cells were provided with chlorodeoxyuridine (CldU, red). 30 min later, cells were washed and supplemented with iododeoxyuridine (IdU, green) and optionally treated with camptothecin (CPT) 50 nM and/or mitomycin C (MMC) 200 nM for 30 min. Green tracks were measured to assess fork speed. (B) Control and PCNA-K164R mouse embryonic fibroblasts (MEFs) were subjected to the DNA fiber protocol described in (A). At least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗p < 0.001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (C) Representative electron microscopy images of reversed (left) or normal (right) replication forks. P, parental strand; D, daughter strand; R, regressed arm. (D) Frequency of reversed forks in the indicated MEFs, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, assessed by EM visualization. Similar results were obtained in two biological replicates and in independent MEF clones (Tables S1A and S1B).
Figure 1 PCNA Ubiquitination Is Required for Replication Fork Slowing and Reversal upon Genotoxic Stress (A) Labeling scheme of DNA fiber experiments: cells were provided with chlorodeoxyuridine (CldU, red). 30 min later, cells were washed and supplemented with iododeoxyuridine (IdU, green) and optionally treated with camptothecin (CPT) 50 nM and/or mitomycin C (MMC) 200 nM for 30 min. Green tracks were measured to assess fork speed. (B) Control and PCNA-K164R mouse embryonic fibroblasts (MEFs) were subjected to the DNA fiber protocol described in (A). At least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗p < 0.001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (C) Representative electron microscopy images of reversed (left) or normal (right) replication forks. P, parental strand; D, daughter strand; R, regressed arm. (D) Frequency of reversed forks in the indicated MEFs, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, assessed by EM visualization. Similar results were obtained in two biological replicates and in independent MEF clones (Tables S1A and S1B).CC BY-NC-ND · PubMed Central
Figure 2 K63-Linked, UBC13-Dependent Polyubiquitination Is Required for Drug-Induced Fork Slowing and Reversal (A) Cells conditionally (+Dox) replacing endogenous ubiquitin with a K63R ubiquitin mutant were subjected to the DNA fiber protocol as in Figure 1A. The ratio between green and red tracts is plotted, to display drug-induced fork slowing. (B) Frequency of replication fork reversal in cells replacing endogenous ubiquitin with WT or K63R-ubiquitin, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, assessed by EM visualization. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S2A). (C) Wild-type (WT) or UBC13-knockout (UBC13-KO) HCT116 cells were subjected to the DNA fiber protocol in Figure 1A. (D) The same cell lines as in (C) were used for DNA fiber analysis, upon optional 5 J/m2 UV-C irradiation in between the two labeling periods. Top right: the western blot shows the absence of UBC13 in UBC13-KO HCT116 cells. GAPDH, loading control. In (A), (C), and (D) at least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (E) Frequency of replication fork reversal in WT and UBC13-KO HCT116 cells, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, or 1 hr after 5 J/m2 UV-C irradiation. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S2B).
Figure 2 K63-Linked, UBC13-Dependent Polyubiquitination Is Required for Drug-Induced Fork Slowing and Reversal (A) Cells conditionally (+Dox) replacing endogenous ubiquitin with a K63R ubiquitin mutant were subjected to the DNA fiber protocol as in Figure 1A. The ratio between green and red tracts is plotted, to display drug-induced fork slowing. (B) Frequency of replication fork reversal in cells replacing endogenous ubiquitin with WT or K63R-ubiquitin, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, assessed by EM visualization. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S2A). (C) Wild-type (WT) or UBC13-knockout (UBC13-KO) HCT116 cells were subjected to the DNA fiber protocol in Figure 1A. (D) The same cell lines as in (C) were used for DNA fiber analysis, upon optional 5 J/m2 UV-C irradiation in between the two labeling periods. Top right: the western blot shows the absence of UBC13 in UBC13-KO HCT116 cells. GAPDH, loading control. In (A), (C), and (D) at least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (E) Frequency of replication fork reversal in WT and UBC13-KO HCT116 cells, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, or 1 hr after 5 J/m2 UV-C irradiation. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S2B).CC BY-NC-ND · PubMed Central
Figure 3 ZRANB3 Is Required for Efficient Replication Fork Slowdown and Fork Reversal upon Different Genotoxic Treatments (A) Wild-type (WT) or ZRANB3-knockout (ZRANB3-KO) U2OS cells were subjected to the DNA fiber protocol as in Figure 1A. (B) The same cell lines as in (A) were used for DNA fiber analysis, upon optional 5 J/m2 UV-C irradiation in between the two labelings. Top right: the western blot shows the absence of ZRANB3 in ZRANB3-KO U2OS cells. β tubulin, loading control. In (A) and (B), at least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.1; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (C) Frequency of replication fork reversal in WT and ZRANB3-KO U2OS cells, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, or 1 hr after 5 J/m2 UV-C irradiation irradiation. In brackets, the number of analyzed molecules are shown. Similar results were obtained in two biological replicates and in two independent ZRANB3-KO clones (Tables S3A and S3B).
Figure 3 ZRANB3 Is Required for Efficient Replication Fork Slowdown and Fork Reversal upon Different Genotoxic Treatments (A) Wild-type (WT) or ZRANB3-knockout (ZRANB3-KO) U2OS cells were subjected to the DNA fiber protocol as in Figure 1A. (B) The same cell lines as in (A) were used for DNA fiber analysis, upon optional 5 J/m2 UV-C irradiation in between the two labelings. Top right: the western blot shows the absence of ZRANB3 in ZRANB3-KO U2OS cells. β tubulin, loading control. In (A) and (B), at least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.1; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (C) Frequency of replication fork reversal in WT and ZRANB3-KO U2OS cells, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM or MMC 200 nM, or 1 hr after 5 J/m2 UV-C irradiation irradiation. In brackets, the number of analyzed molecules are shown. Similar results were obtained in two biological replicates and in two independent ZRANB3-KO clones (Tables S3A and S3B).CC BY-NC-ND · PubMed Central
Figure 4 Fork Progression, Fork Remodeling and Chromosomal Integrity Defects upon Inactivation of Different ZRANB3 Domains (A) Left: schematic representation of ZRANB3 domain organization and of mutations analyzed in this study. Right: western blot analysis of ZRANB3 in the indicated cell lines. (B) The indicated stable cell lines, expressing WT or mutant ZRANB3, were used for DNA fiber analysis as in Figure 1A, upon optional CPT 50 nM treatment. At least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗∗p < 0.0001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (C) Frequency of replication fork reversal in the indicated cell lines, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S4). (D) Left: number of chromosomal abnormalities per indicated cell line, as determined by metaphase spreading upon optional 8 hr CPT treatment (50 nM) and 16 hr nocodazole treatment (200 ng/mL). Error bars, standard deviations. Right: representative DAPI stained metaphase; the arrow points to a chromosome break. Scale bar, 5 μm. Western blot analysis of ZRANB3 protein levels in U2OS cell lines used in (D, left). In (A) and (D), the expression level of HA-tagged ZRANB3 WT and mutant proteins (retarded mobility) is close to endogenous ZRANB3 levels in U2OS cells. β tubulin, loading control.
Figure 4 Fork Progression, Fork Remodeling and Chromosomal Integrity Defects upon Inactivation of Different ZRANB3 Domains (A) Left: schematic representation of ZRANB3 domain organization and of mutations analyzed in this study. Right: western blot analysis of ZRANB3 in the indicated cell lines. (B) The indicated stable cell lines, expressing WT or mutant ZRANB3, were used for DNA fiber analysis as in Figure 1A, upon optional CPT 50 nM treatment. At least one hundred tracts were scored per sample. Whiskers: 10th–90th percentile (∗∗∗∗p < 0.0001; ns, non-significant; Mann-Whitney test). Similar results were obtained in at least two biological replicates. (C) Frequency of replication fork reversal in the indicated cell lines, assessed by EM visualization, upon optional 1 hr treatment with CPT 50 nM. In brackets, the number of analyzed molecules. Similar results were obtained in two biological replicates (Table S4). (D) Left: number of chromosomal abnormalities per indicated cell line, as determined by metaphase spreading upon optional 8 hr CPT treatment (50 nM) and 16 hr nocodazole treatment (200 ng/mL). Error bars, standard deviations. Right: representative DAPI stained metaphase; the arrow points to a chromosome break. Scale bar, 5 μm. Western blot analysis of ZRANB3 protein levels in U2OS cell lines used in (D, left). In (A) and (D), the expression level of HA-tagged ZRANB3 WT and mutant proteins (retarded mobility) is close to endogenous ZRANB3 levels in U2OS cells. β tubulin, loading control.CC BY-NC-ND · PubMed Central

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Authors
Vujanovic, M., Krietsch, J., Raso, M. C., Terraneo, N., Zellweger, R., Schmid, J. A., Taglialatela, A., Huang, J.-W., Holland, C. L., Zwicky, K., Herrador, R., Jacobs, H., Cortez, D., Ciccia, A., Penengo, L., & Lopes, M.
Affiliations
  • University of Zurich (CH)
  • Columbia University Irving Medical Center (US)
  • Vanderbilt University (US)
  • The Netherlands Cancer Institute (NL)
Corresponding author
Lopes, Massimo
ORCID
Venue
Molecular Cell
Publisher
Elsevier BV
Published
2017-09
Volume
67
Issue
5
Pages
882-890.e5
ISSN
1097-2765 (ISSN-L)
DOI
10.1016/j.molcel.2017.08.010
PMC
PMC5594246
OpenAlex
W2751450140
Cited by
227
References
39
Citations by year
2026: 10 · 2025: 19 · 2024: 25 · 2023: 27 · 2022: 23 · 2021: 28 · 2020: 35 · 2019: 26 · 2018: 27 · 2017: 7
FWCI
10.62
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Cell (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DORA PSI (Paul Scherrer Institute) (submittedVersion, cc-by-nc-nd)
Funders
  • SNF (31003A_146924, 31003A_169959, 31003A_166370)
  • ERC Consolidator (617102)
  • Helmut Horten
  • NIH (R01CA197774, GM116616)
  • KWF (NKI-2012-5243)
  • ZonMW Top (91213018)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
Keywords
replication fork reversal, DNA damage tolerance, ZRANB3, DNA translocase, Ubc13, K63-linked polyubiquitin, template switching, chromosome breakage, cancer chemotherapy
MeSH terms
Animals, DNA Damage, DNA Replication, DNA Helicases, DNA, Neoplasm, Humans, Kinetics, Mutation, Neoplasms, Transfection, Replication Origin, Proliferating Cell Nuclear Antigen, Polyubiquitin, RNA Interference, Ubiquitin-Conjugating Enzymes, HCT116 Cells, Mice, Ubiquitination, HEK293 Cells, CRISPR-Cas Systems

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