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DNA interstrand cross-link repair requires replication-fork convergence

Nature Structural & Molecular Biology volume 22, pages 242–247 (2015)Cite this article

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Abstract

DNA interstrand cross-links (ICLs) prevent strand separation during DNA replication and transcription and therefore are extremely cytotoxic. In metazoans, a major pathway of ICL repair is coupled to DNA replication, and it requires the Fanconi anemia pathway. In most current models, collision of a single DNA replication fork with an ICL is sufficient to initiate repair. In contrast, we show here that in Xenopus egg extracts two DNA replication forks must converge on an ICL to trigger repair. When only one fork reaches the ICL, the replicative CMG helicase fails to unload from the stalled fork, and repair is blocked. Arrival of a second fork, even when substantially delayed, rescues repair. We conclude that ICL repair requires a replication-induced X-shaped DNA structure surrounding the lesion, and we speculate on how this requirement helps maintain genomic stability in S phase.

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Figure 1: A single fork stalled at an ICL fails to undergo approach.
Figure 2: CMG is not evicted from a single fork stalled at an ICL.
Figure 3: A single stalled fork does not undergo incisions.
Figure 4: The order of replisome arrival at an ICL does not determine which leading strand undergoes lesion bypass.

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Acknowledgements

We thank S. Elledge, L. Zou, A. Smogorzewska, P. Knipscheer and the members of the Walter laboratory for feedback on the manuscript. M.B. was supported by Human Frontiers Science Program long-term fellowship LT000773/2010-l and European Molecular Biology Organization long-term fellowship ALTF 742-2009. A.M. was supported by a Natural Sciences and Engineering Research Council of Canada scholarship. M.A.C. was supported by UK Royal Society grant UF100717 and Fell Fund grant 103/789. J.C.W. was supported by US National Institutes of Health grants GM62267 and HL098316. J.C.W. is supported as an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Jieqiong Zhang, James M Dewar & Magda Budzowska

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Anna Motnenko & Martin A Cohn

  3. Department of Biological Chemistry and Molecular Pharmacology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Johannes C Walter

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  1. Jieqiong Zhang
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Contributions

J.M.D. generated the lacO array (48 lacO repeats) and validated its use as a replication-fork barrier; M.B. and J.Z. prepared pICL-lacOPt; A.M. and M.A.C. prepared psoralen-cross-linked oligonucleotides; J.Z. and J.C.W. designed and analyzed the experiments; J.Z. performed all the experiments; J.Z. and J.C.W. prepared the manuscript.

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Correspondence to Johannes C Walter.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Model for ICL repair with two forks and one fork.

Model for ICL repair with two forks (a) and one fork (b). The details of the single fork model are inferred from the mechanism previously established for the fork convergence modeln6–11.

Supplementary Figure 2 The LacI-lacO array efficiently blocks fork progression.

(a) Schematic of placO replication intermediates expected in the presence and absence of LacI after digestion with XmnI.

(b) placO was replicated with or without LacI, and IPTG was added at the indicated times. Replication intermediates were digested with XmnI, separated on an agarose gel, and detected by autoradiography.

Supplementary Figure 3 Leading strands persist at –20 to –40 when there is only a single fork.

(a) The intensity of leading strands located between the –20 to –40 positions in Fig. 1d and two repetitions of this experiment was quantified and graphed. Error bars represent standard deviations.

(b) Schematic illustration of nascent leading strands generated in the experiment described in (c).

(c) The LacI-lacO array does not inhibit ICL repair once forks have converged on the ICL. pICL-lacO was replicated in egg extracts, and LacI was added at the indicated times relative to NPE addition. Nascent leading strands were digested with AflIII and EcoRI and analyzed on a sequencing gel, as described in Fig. 1d. Note that LacI addition at 0 and 8 minutes after replication initiation, when most forks had not yet converged on the ICL (lane 11), inhibited approach (lanes 6–10 and 12–16). In contrast, LacI addition at 13 and 30 minutes, when most forks had converged (lanes 17 and 23), was not inhibitory for approach (lanes 18–22 and 24–27). Asterisk (*), background bands described in (d).

(d) The LacI-lacO array does not inhibit the approach of pICL repair in trans. pICL (which lacks the lacO array) and placO (no ICL) were mixed and replicated in the presence of buffer or LacI, as indicated. Samples were digested with AflIII, and leading strands of pICL were monitored on a sequencing gel. A series of species differing in size by multiples of 30 nt was observed whenever a lacO-containing plasmid was replicated in the presence of LacI (* bands in the right panel, see also Fig. 1d, 1f; Supplementary Figure. 3c, 4b). The formation of the ladder was independent of restriction enzyme digestion (data not shown), indicating that non-ligated nascent strands generated within the lacO array give rise to the repeating pattern.

Supplementary Figure 4 A single fork remains competent for ICL repair after prolonged stalling.

(a) Schematic illustration of nascent leading strands generated in the presence and absence of LacI.

(b) pICL-lacO was replicated with or without LacI, and IPTG was added at the indicated times. Nascent leading strands were digested with AflIII and EcoRI and analyzed on a sequencing gel, as described in Fig. 1d. Asterisk (*), background bands described in Supplementary Fig. 3d.

(c) The intensity of the –20 to –40 products after IPTG addition in (b) was quantified and graphed.

Supplementary Figure 5 ChIP results and quantification of incision assays.

(a-c) ChIP results for MCM7, CDC45, FANCD2, XPF, and SLX4

(a) Experimental replicate of the MCM7 and CDC45 ChIP described in Fig. 2b.

(b) Two independent examples of FANCD2 ChIP. pICL-lacO and pQuant were replicated with or without LacI, and IPTG was added immediately before the 20 minute time point, as indicated (green arrow). At different times, samples were withdrawn for FANCD2 ChIP using primer pairs for the ICL locus (Fig. 2a) or pQuant (Ctrl).

(c) Experimental replicate of the XPF and SLX4 ChIP described in Fig. 3e.

(d) Quantification of Incision Assay

We quantified and graphed the efficiency of incision in the experiment shown in Fig. 3d (left) and in two repetitions of this experiment (middle and right). Incisions convert the X-shaped parental structure into linear products (Fig. 3c, left). However, linear products are created not only via incision, but also from nascent strands generated during lesion bypass, from homologous recombination, and from replication of undamaged plasmid. In contrast, the disappearance of X-shaped molecules is caused exclusively by incision, making the reduction in the intensity of the X-shaped band a better readout of incision. To compare incisions in the presence of one and two forks, we therefore determined the reduction of the X-shaped species in the presence of buffer (two forks), in the presence of LacI (one fork), and in the presence of LacI and IPTG (two forks). We found that in the three experiments shown above, LacI inhibited the reduction in X-shaped species by an average of ~70%, consistent with the fact that on 26% of molecules, a leftward replication fork reaches the ICL even in the presence of LacI (Fig. 1d).

Supplementary Figure 6 Fork convergence versus traverse.

When a single DNA replication fork encounters an ICL, it can pause until the arrival of a second fork (left arrow, “fork convergence”) or bypass the lesion (right arrow, “traverse”)22. In both cases, an X-shaped DNA structure surrounding the ICL is generated (grey box). If traverse happened in our system, the leading strands of the rightward, single fork should approach to –1 (orange strand) since CMG either dissociates or bypasses the ICL, yet they stall at the –20 position. The 5′ ends of the leading strands of the traversed fork might be heterogeneous, depending on where DNA synthesis restarts on the other side of the ICL (blue strand). In addition, lagging strands of the traversed fork should approach directly to the lesion without –20 stalling since there is no CMG travelling ahead of the polymerase (purple strand). Although we observed a low abundance of nascent strands between the ICL and the lacO array, they initially stalled at the –20 position, after which they underwent approach (Fig. 1d, 1f, leftward fork panel). Therefore, we conclude that these signals came from the arrival of the leftward forks (green strand; Supplementary Figure. 2) rather than traverse (purple strand).

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Zhang, J., Dewar, J., Budzowska, M. et al. DNA interstrand cross-link repair requires replication-fork convergence. Nat Struct Mol Biol 22, 242–247 (2015). https://doi.org/10.1038/nsmb.2956

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