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Replication fork uncoupling causes nascent strand degradation and fork reversal

Nature Structural & Molecular Biology volume 30, pages 115–124 (2023)Cite this article

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Abstract

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts and validated this approach with experiments in human cells. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. Notably, the replicative helicase remains bound during NSD and fork reversal. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and elucidate key events that precede fork reversal.

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Fig. 1: Induction of synchronous and localized NSD in vitro.
Fig. 2: NSD involves DNA2 but not MRE11.
Fig. 3: NSD involves fork reversal.
Fig. 4: Uncoupling promotes NSD and fork reversal.
Fig. 5: The replisome remains associated with DNA during NSD.
Fig. 6: DNA2 degrades replication forks and reversed forks.
Fig. 7: Model for NSD in Xenopus egg extracts.

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Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH grant R35GM128696 to J.M.D., ACS grant IRG-15-169-56 to J.M.D., funding provided by the Breast Cancer Research Foundation to D.C. and Vanderbilt-Ingram Cancer Center Support Grant P30CA068485. We thank V. Costanzo (IFOM, FIRC Institute for Molecular Oncology, Italy and University of Milan, Italy) for antibodies to Xenopus SAMHD1 and SMARCAL1 as well as for helpful discussion about the work. We thank J. Walter (Harvard Medical School, USA and Howard Hughes Medical Institute, USA) for antibody to Xenopus FAN1.

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Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA

    Tamar Kavlashvili, Wenpeng Liu, Taha M. Mohamed, David Cortez & James M. Dewar

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  1. Tamar Kavlashvili
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  2. Wenpeng Liu
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  3. Taha M. Mohamed
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Contributions

W.L. performed the experiments in Figs. 2d and 6h and Extended Data Figs. 1b, 3j,k, 4f and 8f. T.M.M. performed the experiments in Fig. 3h and Extended Data Fig. 1c. T.K. performed all other experiments. T.K. and J.M.D. designed the experiments, analyzed the data and wrote the paper with input from D.C., T.M.M. and W.L.

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Correspondence to James M. Dewar.

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Nature Structural & Molecular Biology thanks Stephan Hamperl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Beth Moorefield, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Nascent strand degradation is an initial response to fork stalling.

(A) A current model for fork reversal and nascent strand degradation. Note that fork reversal enzymes are thought to directly convert reversed forks back to replication forks1,2,3. (B) U2OS cells were pulse-labeled with CldU then IdU followed by treatment with the indicated concentrations of aphidicolin so that loss of the IdU labeled tracks could be monitored as a measurement of NSD. DNA fiber analysis was performed to determine the lengths of CldU and IdU labeled DNA tracks. n indicates the number of DNA fibers measured for each condition. P-values from a one-way ANOVA performed using Dunnett’s multiple comparisons test are reported for each condition. Non-significant comparisons are indicated as ‘ns’. Although lower concentrations of aphidicolin (0.2–1 µM) did not impact the IdU/CldU ratio higher concentrations (4–10 µM) significantly reduced the IdU/CldU ratio indicating that NSD took place. Thus, we observed a dose-dependent induction of NSD by aphidicolin. (C) U2OS cells were pulse labeled with CldU and IdU, then treated with the indicated concentrations of aphidicolin and hydroxyurea (HU). DNA fiber analysis was performed to determine the lengths of CldU and IdU labeled DNA tracks. n indicates the number of DNA fibers measured for each condition. P-values from a one-way ANOVA performed using Dunnett’s multiple comparisons test are reported for each condition. Non-significant comparisons are indicated as ‘ns’. Co-addition of the RAD51 inhibitor B02 (RAD51-i)4 was also performed as a positive control for degradation in the presence of HU. RAD51-i addition inactivated fork protection and led to extensive degradation in the presence of HU, as previously described5. In contrast, 1, 4, and 8 mM HU did not result in detectable NSD, consistent with limited degradation in the presence of HU alone6. Notably, the extent of NSD following 10 µM aphidicolin treatment was comparable to the level observed following HU treatment and RAD51-i addition. (D) To stall replication forks in vitro, plasmid DNA was replicated using Xenopus egg extracts and newly-synthesized nascent strands were radiolabeled by inclusion of [α-32P]dATP. In this system, plasmid templates replicate semi-synchronously from a single origin per plasmid7. 6 minutes after initiation, reactions were treated with aphidicolin, to stall DNA synthesis, or vehicle control. As a loading control (Ctrl) the reactions include a smaller plasmid that was radiolabeled prior to the experiment and did not undergo replication. (E) DNA structures from (d) were purified and digested with XmnI, which cuts the plasmid once. (F) DNA taken from (e) at the moment of aphidicolin addition (6 minutes after initiation) was separated by 2-D gel electrophoresis and visualized by autoradiography. The entire Double-Y and Bubble arcs (i) were visible (ii), indicating that DNA molecules of all sizes, and thus at all stages of replication, were present. Note that origin firing generates bubble structures, which are converted to double-Ys once one of the two forks moves beyond the restriction site (iii). (G) Samples from (e) were separated on an agarose gel and visualized by autoradiography. Time after aphidicolin addition is indicated. (H) Quantification of DNA synthesis from (g) normalized to the maximum signal across all time points and conditions. Mean ± S.D., n = 3 independent experiments. (I) Quantification of DNA synthesis in (g) normalized to the maximum signal for each condition across all time points. Mean ± S.D., n = 3 independent experiments. Following vehicle treatment in (h)-(i), radioactive signal increased and then plateaued, indicating that a single round of replication was completed. In contrast, following aphidicolin treatment in (h)-(i) signal declined, indicating that degradation took place. Importantly, signal was appreciably reduced by 60 minutes, indicating that degradation was an initial response and did not require prolonged fork stalling. (J) To test whether degradation in (h)-(i) corresponded to both strands or nascent strands only, pre-radiolabeled plasmid DNA was replicated using Xenopus egg extracts. 6 minutes after initiation, DNA synthesis was inhibited by the addition of Aphidicolin, as in (e). (K) XmnI digested molecules were separated on an agarose gel and visualized by autoradiography. As a loading control (Ctrl) the reactions include a smaller radiolabeled plasmid that did not undergo replication. (L) Quantification of (k) as in (h). Mean ± S.D., n = 3 independent experiments. (M) Quantification of (k) as in (i). Mean ± S.D., n = 3 independent experiments. In (l)-(m) parental strand signal was relatively stable and there was little difference between aphidicolin and vehicle treatment. Moreover, there was no appreciable loss of parental strand signal for the first 120 minutes, during which time most of the nascent strands were degraded in (h)-(i). Thus, most signal loss was due to degradation of nascent DNA strands. Overall, (d)-(l) show that degradation of nascent strands is an initial response to fork stalling.

Source data

Extended Data Fig. 2 Characterization of localized, synchronous nascent strand degradation.

(A) Cartoon depicting the source of θ* structures in Fig. 1B. The XmnI site is located 1013 base pairs away from the closest edge of the lacO array. (B) DNA structures from Fig. 1A were treated with human topoisomerase II (hTop2) or buffer control, then separated on an agarose gel and visualized by autoradiography. hTop2 treatment converted θ* signal back to θs, demonstrating that θ* structures are topoisomers of θs. Note that in lane 4 an additional band appears below the θ, suggesting that replication forks undergo remodeling (see Fig. 3 and related discussion in the main text). (C) Quantification of Replication Intermediates (RIs) as a % of total lane signal from Fig. 1E. Mean ± S.D., n = 5 independent experiments. (D) Quantification of total lane signal from Fig. 1E. Mean ± S.D., n = 5 independent experiments. (E) Plasmid DNA harboring a 32xlacO array (p[lacO]) or a 50xlacO array (p[lacOx50]) was incubated with LacR then replicated in Xenopus egg extracts. Once forks were localized to the LacR barrier, NSD was induced by addition of IPTG and aphidicolin. Purified DNA was subjected to restriction digest so that replication fork structures (RIs) could be visualized. (F) Samples from (e) were separated on an agarose gel and visualized by autoradiography. Note that a mobility shift can readily be detected for p[lacOx50], suggesting that fork remodeling occurs (see Fig. 4 and related main text). (G) Quantification of the amount of DNA degraded in (f). Normalized degradation accounts for the different backbone sizes of the 50x and 32x lacO plasmids. Mean ± S.D., n = 3 independent experiments. The 50xlacO array is ~60% larger than the 32xlacO array so if NSD was influenced by the proximity of the replication forks on either side of the lacO array there would be a substantial difference in NSD between p[lacO] and p[lacOx50]. However, the difference in normalized degradation between the two plasmids is negligible. Thus, NSD does not appear to be influenced by the proximity of the two converging forks.

Source data

Extended Data Fig. 3 Characterization of MRE11 and DNA2 activity during nascent strand degradation.

(A) Cartoon depicting the roles of MRE11 and DNA2 in resection at double-strand breaks. (B) Cartoon depicting the roles of MRE11 and DNA2 in resection at reversed forks. (C) Linear radiolabeled DNA was incubated in Xenopus egg extracts in the presence of MRE11 inhibitor (MRE11-i) or DNA2 inhibitor (DNA2-i) or both. Double-Strand Break (DSB) resection involves both enzymes in Xenopus egg extracts (b) and is blocked by inactivation of both DNA2 and MRE118. Thus, if both MRE11-i and DNA2-i effectively inhibit their targets then combination of both drugs should block resection. (D) Samples from (c) were separated on an agarose gel and visualized by autoradiography. In the vehicle control the linear substrate was rapidly degraded (lanes 1-4). We also observed a small number of additional products that arose from end-joining (EJ(L) and EJ(C)). As expected9, DNA2-i and MRE11-i each inhibited resection (lanes 5-8, 9-12), while a combination of both inhibitors almost completely blocked resection (lanes 13-16). (E) Quantification of total signal from (d). Mean ± S.D., n = 3 independent experiments. (F) Depiction of the DNA structures generated by Fig. 2A, prior to restriction digest. (G) DNA intermediates from (f) were separated on an agarose gel and visualized by autoradiography. Uncoupled species (θ*) are indicated. (H) Quantification of total lane signal from Fig. 2B. Mean ± S.D., n = 3 independent experiments. (I) Quantification of RI signal in Fig. 2B as a percentage of total signal for each lane. Mean ± S.D., n = 3 independent experiments. (J) U2OS cells were pulse labeled with CldU and IdU, then treated with either aphidicolin, or hydroxyurea combined with RAD51-i. MRE11-i, DNA2-i, or vehicle control were also added, as indicated. DNA fiber analysis was performed to determine the lengths of IdU and CldU labeled DNA tracks. n indicates the number of DNA fibers measured for each condition. P-values from a one-way ANOVA performed using Dunnett’s multiple comparisons test are reported for each condition. Non-significant comparisons are indicated as ‘ns’. Treatment with Rad51-i compromised fork protection and led to NSD in the presence of HU, as previously reported5. Additionally, NSD was rescued by either DNA2-i or MRE11-i, as expected10, confirming that both drugs were able to effectively inhibit their targets. Aphidicolin treatment resulted in NSD (as in Extended Data Fig. 1B) and this was rescued by DNA2-i but not MRE11-i. Thus, NSD caused by aphidicolin treatment results in DNA2-dependent but MRE11-independent degradation. (K) HCT116 cells were transfected with the indicated siRNA. 72 hours later cells were pulse labeled with CldU and IdU, then treated with aphidicolin. DNA fiber analysis was performed to determine the lengths of IdU and CldU labeled DNA tracks. n indicates the number of DNA fibers measured for each condition. P-values from a one-way ANOVA performed using Dunnett’s multiple comparisons test are reported for each condition. Non-significant comparisons are indicated as ‘ns’.

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Extended Data Fig. 4 SAMHD1 and FAN1 are not involved in nascent strand degradation.

(A) Forks were localized to a LacR barrier and NSD was induced by addition of IPTG and aphidicolin in SAMHD1- and FAN1-immunodepleted Xenopus egg extracts. Purified DNA was subjected to restriction digest so that replication fork structures could be visualized (RIs). (B) Immunodepleted extracts and the corresponding immunoprecipitates (beads) from (a) were analyzed by Western blotting to determine the extent of SAMHD1 and FAN1 immunodepletion. In both cases the immunodepletion went to completion because no detectable protein was recovered by round 3 (compare lanes 7-8 to lane 9). Additionally, at least 95% of protein was removed in each case (compare lanes 1 and 5). (C) Samples from (a) were separated on an agarose gel and visualized by autoradiography. (D) Quantification of RI signal from (c). (E) Quantification of RI signal from an experimental replicate of (c). In both (d) and (e) neither SAMHD1 nor FAN1 depletion altered the rate of degradation, indicating that they do not participate in NSD. (F) U2OS cells were transfected with the indicated siRNA, pulse labeled with CldU and IdU, then treated with aphidicolin. DNA fiber analysis was performed to determine the lengths of CldU and IdU labeled DNA tracks. n indicates the number of DNA fibers measured for each condition. P-values from a one-way ANOVA performed using Dunnett’s multiple comparisons test are reported for each condition. Non-significant comparisons are indicated as ‘ns’. Neither SAMHD1 nor FAN1 affected degradation, consistent with (d)-(e).

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Extended Data Fig. 5 Characterization of DNA structures detected by 2-D gel during nascent strand degradation.

(A) Quantification of Double-Ys from Fig. 3C expressed as a percentage of total signal at T = 0. Mean ± S.D., n = 3 independent experiments. (B) Quantification of remodeled forks from Fig. 3C expressed as a percentage of total signal at T = 0. Mean ± S.D., n = 3 independent experiments. (C) Cartoon indicating the different DNA structures that the remodeled forks in Fig. 3C could correspond to. (D) Expected 2-D gel migration pattern of the structures depicted in (c). The region highlighted in red indicates where each structure is expected to migrate. ‘?’ indicates structures whose expected migration on a 2-D gel is unclear. The remodeled forks (Fig. 3C) did not correspond to canonical Holliday junctions (i) or hemicatenanes (ii). However, these species could have reflected reversed forks (iii), hemicatenanes (iv), or Holliday junctions (v) in the wake of one or both forks. (E) Samples from Fig. 3C,iii were treated with buffer or RuvC, which cleaves reversed forks and Holliday junctions, but not hemicatenanes or other replication fork structures11. RuvC-treated samples were then separated by 2-D gel electrophoresis and visualized by autoradiography. (F) Quantification of DYs and RFs following RuvC treatment in (e) expressed relative to the abundance of each structure in the buffer treated condition. RuvC treatment had essentially no effect on Double-Ys, as expected, but reduced remodeled forks by ~5-fold. Thus, the remodeled forks contain four-way junctions that arise from reversed forks or Holliday junctions that is D-loops. (G) Quantification of RFs and DYs from (c) expressed relative to total signal on each gel. (H) Cartoon indicating the structure of a D-loop and a reversed fork. (I) Expected 2-D gel migration pattern of the structures depicted in (h). The region highlighted in red indicates where each structure is expected to migrate. (J) Cartoon indicating the restriction digest strategy used to excise individual replication forks for 2-D gel analysis. The DraIII site is 450 bp away from the nearest edge of the lacO array and 1250 bp away from the XhoI site.(K) Forks were localized to a LacR barrier and NSD was induced by addition of IPTG and aphidicolin in SMARCAL1-immunodepleted Xenopus egg extracts. Purified DNA was subjected to restriction digest so that replication fork structures could be visualized (RIs). (L) Immunodepleted extracts and the corresponding immunoprecipitates (beads) from (k) were analyzed by Western blotting to determine the extent of SMARCAL1 immunodepletion. Immunoblotting was performed using an antibody generated by this study (i) and the previously published antibody (ii)12. No detectable protein was recovered by the final round of depletion (compare lanes 7-8 to lane 9), indicating that the depletion was successful. Additionally, at least 99% of protein was removed (compare lanes 1 and 6 for (ii).(M) Samples from (k) were separated on an agarose gel and visualized by autoradiography. (N) Quantification of RI signal from (m). (O) Quantification of RI signal from an experimental replicate of (m). In both (n) and (o) SMARCAL1 immunodepletion had no impact on degradation.

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Extended Data Fig. 6 Characterization of stalled and uncoupled replication forks.

(A) Cartoon depicting the strategy to test the role of helicase stalling during NSD. If helicase stalling triggers NSD then NSD should be increased in the absence of IPTG. If uncoupling triggers NSD then addition of IPTG should increase NSD. (B) Depiction of the DNA structures generated by Fig. 4A, prior to restriction digest. (C) Samples from (e) were separated on an agarose gel and visualized by autoradiography. θ* species arise from uncoupling. θ- species arise from the low level of uncoupling that occurs even in the presence of LacR, which is not a complete block to helicase progression13. (D) Depiction of different models for degradation of leading and lagging strands during NSD observed in Fig. 4 and the expected effect on degradation of LWS and RWS depicted in Fig. 4A. In (i) both leading and lagging strand are degraded simultaneously and synchronously, which should result in disappearance of RWS before LWS. In (ii) lagging strands only are degraded synchronously, which should result in persistence of ~50% of RWS and LWS until the leading strand are degraded by nuclease activity that initiated at lagging strands of the opposite fork. This would result in biphasic kinetics of degradation. In (iii) lagging strands only are degraded asynchronously, which should result in loss of RWS and LWS at the same rate. (E) Uncropped gel of Fig. 4B lanes 1-4 shown after a longer exposure so that the weaker rightward strands (RWS) could be visualized. (F) Quantification of LWS and RWS from (b). RWS are degraded before LWS, with no evidence of biphasic kinetics, indicating that both strands are degraded simultaneously, as in (a,i). Mean ± S.D., n = 3 independent experiments. (G) NSD was induced as in Fig. 4A in the presence or absence of DNA2-i. (H) Samples from (g) were separated on a native agarose gel and visualized by autoradiography. (I) Quantification of RI signal from (h). Mean ± S.D., n = 3 independent experiments. (J) Quantification of reversed forks from Fig. 4E expressed as a percentage of total signal at T = 0. Mean ± S.D., n = 3 independent experiments.

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Extended Data Fig. 7 Binding of replication fork proteins during NSD.

(A) Cartoon depicting two different models of CMG helicase behavior during fork reversal and NSD. In (i) the CMG helicase translocates onto double-stranded DNA, as suggested1,14, which would result in its removal from DNA15,16,17,18. In (ii) the replisome remains on DNA, suggesting that it resides in a ssDNA bubble ahead of the reversed fork. (B) In parallel to Fig. 5A chromatin bound proteins were analyzed in the presence of DNA2-i. RPA was detected by Western Blotting. (C) Quantification of RPA signal from Fig. 5B. and (b). Mean ± S.D., n = 3 independent experiments. Between 0 and 60 minutes, when most fork reversal took place (Fig. 3D), most RPA signal was due to DNA2 activity. Thus, most RPA signal during this time was due to DNA2-dependent NSD, which may mask any dissociation of RPA that occurred due to reannealing of parental DNA strands.

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Extended Data Fig. 8 Additional degradation steps during NSD.

(A) Cartoon of different models to explain a role for DNA2 in degrading Y-shaped forks during NSD and the expected impact of loss of DNA2 activity on Y-shaped forks in each case. (i) depicts a model where DNA2 degrades reversed forks to generate Y-shaped forks. In this model the Y-shaped forks are degraded as consequence of prior DNA2 activity at reversed forks. (ii) depicts the impact of impaired DNA2 activity on (i). Y-shaped forks should decrease in abundance as less degradation of reversed forks should reduce the formation of Y-shaped forks. (iii) depicts a model where DNA2 degrades forks prior to fork reversal. (iv) depicts the impact of impaired DNA2 activity on (iii). Y-shaped forks should increase in abundance due to less degradation of nascent strands, which is the source of the radioactive signal that is measured. (B) Quantification of Double-Ys from Fig. 6B expressed as a percentage of total replication intermediates (RIs). Mean ± S.D., n = 3 independent experiments. (C) Quantification of reversed forks from Fig. 6B expressed as a percentage of total replication intermediates (RIs). Mean ± S.D., n = 3 independent experiments. (D) U2OS cells treated with control or RAD51 siRNA were lysed and analyzed by western blotting for RAD51. Total protein levels were also determined by stain-free imaging (Bio-Rad). (E) HCT116 cells were transfected with RAD51 siRNA, pulse labeled with CldU and IdU, and then treated with aphidicolin. DNA fiber analysis was performed to determine the lengths of CldU and IdU labeled DNA tracks in each condition. (F) HCT116 cells were transfected with control or RAD51 siRNA, pulse labeled with CldU and IdU, then treated with aphidicolin. DNA fiber analysis was performed to determine the lengths of the CldU and IdU labeled DNA tracks. n indicates the number of DNA fibers measured for each condition. P-values from a one-way ANOVA performed using Dunnett’s multiple comparisons test are reported for each condition. Non-significant comparisons are indicated as ‘ns’. Degradation still occurred even following knockdown of RAD51, consistent with results from U2OS cells in Fig. 6H. (G) Potential models for template switching, based on our new model for NSD (Fig. 7). (i) After encountering a polymerase-blocking lesion (yellow) replication forks uncouple, resulting in a stalled replisome (red). (ii) The replisome is retained on DNA and parental DNA strands anneal to create a second replication fork that is the substrate for fork reversal. This places the replisome within a single-stranded DNA bubble. (iii) The nascent lagging strands serve as template for leading strand synthesis (green). (iv) Exonuclease activity at the extruded DNA end generates a 3′ overhang. (v) restoration of the reversed fork results in a lagging strand gap that allows loading of a 5′-3′ single-stranded DNA helicase that can unwind the re-annealed parental strands. (vi) nascent strands are extended to the replisome, which resumes unwinding (green). (vii) Without exonuclease activity at the extruded DNA end (in (iv)) restoration of the reversed fork would result in a 3-way fully duplex fork structure that could not be readily unwound. (viii) An alternative model of the response replication fork uncoupling by a polymerase-blocking lesion. (ix) Nascent DNA strands are unwound by single-stranded DNA helicases, allowing them to anneal together. (x) The annealed duplex generated in (ix) is the substrate for fork reversal enzymes, which reanneal parental duplex as the nascent DNA strands are extruded. (xi) Exonuclease activity at the extruded end generates a 3′ overhang, as in (iv), but the bubble structure is more extensive and the parental duplex is more limited. (xii) Restoration of the reversed fork removes all parental duplex, provided that only one of the two strands remains intact. (xiii) nascent strands are extended to the replisome, which resumes unwinding.

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Supplementary information

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Supplementary Note

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Kavlashvili, T., Liu, W., Mohamed, T.M. et al. Replication fork uncoupling causes nascent strand degradation and fork reversal. Nat Struct Mol Biol 30, 115–124 (2023). https://doi.org/10.1038/s41594-022-00871-y

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