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Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis

Nature Structural & Molecular Biology volume 29, pages 932–941 (2022)Cite this article

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Abstract

Processivity clamps tether DNA polymerases to DNA, allowing their access to the primer–template junction. In addition to DNA replication, DNA polymerases also participate in various genome maintenance activities, including translesion synthesis (TLS). However, owing to the error-prone nature of TLS polymerases, their association with clamps must be tightly regulated. Here we show that fork-associated ssDNA-binding protein (SSB) selectively enriches the bacterial TLS polymerase Pol IV at stalled replication forks. This enrichment enables Pol IV to associate with the processivity clamp and is required for TLS on both the leading and lagging strands. In contrast, clamp-interacting proteins (CLIPs) lacking SSB binding are spatially segregated from the replication fork, minimally interfering with Pol IV-mediated TLS. We propose that stalling-dependent structural changes within clusters of fork-associated SSB establish hierarchical access to the processivity clamp. This mechanism prioritizes a subset of CLIPs with SSB-binding activity and facilitates their exchange at the replication fork.

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Fig. 1: An SSB-binding mutation within Pol IV.
Fig. 2: SSB promotes access of Pol IV to stalled replication forks.
Fig. 3: SSB enriches Pol IV at lesion-stalled replication forks.
Fig. 4: Enrichment of Pol IV at stalled forks promotes TLS.
Fig. 5: SSB-driven ectopic localization of Pol IV or Pol I.
Fig. 6: Roles of SSB in pathway choice and fork compartmentalization.

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Data availability

The data sets from the current study are available from the corresponding author on reasonable request. A structure of E. coli Pol IV was retrieved from the PDB with accession code 4Q43. Source data are provided with this paper.

Code availability

The custom-written computer codes from the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank K. Arnett (Center for Macromolecular Interactions, Harvard Medical School) for training and assistance in using a CD spectropolarimeter, J. Keck (University of Wisconsin, Madison) for providing the fluorescein-conjugated SSB-Ct peptide, D. Li (University of Rhode Island) for providing the N2-FFdG-containing oligomer, J. Kath (Harvard Medical School) for construction of tetracycline-inducible strains, and S. Jergic (University of Wollongong) for providing the various purified Pol III polymerase complexes and helpful discussions. This work was supported by National Institutes of Health grants R01 GM114065 (to J.J.L.), The William F. Milton Fund (to S.C.), F32 GM113516 (to E.S.T.), and Agence Nationale de la Recherche (ANR) Grant (GenoBlock ANR-14-CE09-0010-01) (to V.P.).

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Author notes

  1. Elizabeth S. Thrall

    Present address: Department of Chemistry, Fordham University, New York City, NY, USA

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Seungwoo Chang, Elizabeth S. Thrall, Sadie C. Piatt & Joseph J. Loparo

  2. CRCM (Cancer Research Center of Marseille): Team DNA Damage and Genome Instability, Aix-Marseille University, CNRS, INSERM, Institut Paoli-Calmettes, Marseille, France

    Luisa Laureti & Vincent Pagès

  3. Harvard Graduate Program in Biophysics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Sadie C. Piatt

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Contributions

S.C., E.S.T., L.L., S.C.P., V.P., and J.J.L. designed and performed research. S.C., E.S.T., L L., S.C.P., V.P., and J.J.L. analyzed data. S.C., E.S.T., L.L., V.P., and J.J.L. wrote the paper.

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Correspondence to Joseph J. Loparo.

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Extended data

Extended Data Fig. 1 An SSB-binding defective mutant Pol IV.

a. Formation of nucleoprotein complexes between SSB4 or SSBΔF4 and ssDNA. (Top) An FP-based binding assay scheme. (Bottom) Interaction of T71-FL with SSB4 or SSBΔF4 was measured by changes in FP. Means ± ranges of duplicate measurements. Similar results were reproduced twice. b. Over-expression-induced cell death by RecQ, Exonuclease I, Topoisomerase III, Pol II, or their variants. R503A of RecQ, and R418A, R316A, and Q311A of Exonuclease I, SSB-binding mutations; R448A of RecQ, a negative control mutation; flag, N-terminal FLAG epitope; exoI, topoIII, and polB, coding sequences for exonuclease I, topoisomerase III, and Pol II respectively. c. Location of T120 within Pol IV. (Left) A front view showing the location of T120 relative to the catalytic cleft (yellow arrow). (Right) A side view showing the location of T120 relative to the C-terminal end (P341). C-terminal unstructured region (a.a. 342-351) containing the clamp interacting motif (a.a. 346-351) is missing in the structure. A crystal structure of E. coli Pol IV (PDB 4q43) is modified.

Source data

Extended Data Fig. 2 Fates of stalled replication and associated replication products in our rolling circle replication-based assays.

In these rolling circle DNA templates, the lesion-containing closed circular DNA is the template for leading strand synthesis. During rolling circle DNA replication, the growing leading strand serve as the template for lagging strand synthesis.

Extended Data Fig. 3 Pol IV must bind to the β2 clamp to mediate TLS at the fork.

The N2-FFdG-containing template was replicated in the presence of indicated concentrations of Pol IV or Pol IVΔC6 as shown in Fig. 2a. Pol IVΔC6, a mutant Pol IV lacking the C-terminal clamp binding motif.

Source data

Extended Data Fig. 4 Monitoring resolution pathway choice in cells.

a. Clamp binding motif (CBM) of the ε subunit of the Pol III core complex (αεθ). Shown are wild-type and mutant CBM sequences and their binding affinities to the β2 clamp. ε-cleft, the binding site on the β2 clamp for the CBM of the ε subunit; εQ, CBM with a weakening mutation; εL, CBM with a strengthening mutation. dnaQ(εQ), weakening mutation. θ subunit is omitted in the schematic diagram for simplicity. b. (Left) Site-specific insertion of a single N2-FFdG adduct into the E. coli genome in either the leading or lagging strand template. (Right) Resolution of lesion-stalled replication through TLS leads to the formation of blue-sectored colonies. White colonies represent resolution through DA. c. Deletion of lafU, which is replaced with an frt-kan-frt cassette as a dinB-linked marker for P1 transduction, does not influence pathway choice of N2-FFdG-stalled replisomes in cells. In all ΔlafU-containing strains, the kan cassette was flipped out. Means ± SDs (n = 6 for lafU+; n = 3 for ΔlafU; n, number of independent experiments). d. TLS over N2-FFdG in cells is primarily mediated by Pol IV. Deleting dinB reduces the resolution of N2-FFdG-stalled replisomes through TLS from ~60 to ~30%. Additional deletion of both polB and umuDC does not further reduce resolution through TLS. The residual TLS (~30%) in the ΔdinB ΔpolB ΔumuDC background is presumably mediated by Pol III. However, as Pol IV outcompetes Pol III in mediating TLS over N2-FFdG, the actual contribution of Pol III to TLS in the dinB+ background is likely less than estimated in the ΔdinB background. Ld, leading strand lesion; Lg, lagging strand lesion. Means ± SDs (n = 3; n, number of independent experiments). The statistical significance of the difference between the indicated pairs was determined by two-tailed Welch’s t-test; numbers, p values. e. Weakening the SSB-Pol IV interaction increases the MMS-induced SOS response. (Left) Resolution of stalled replication through repriming results in induction of the SOS damage response. TLS at the fork inhibits repriming. (Right) The MMS-induced SOS response in strains bearing indicated dinB alleles. The SOS reporter strains express green fluorescent protein (GFP) from a genomic expression cassette, in which the transcription of gfp is under the control of the sulA promoter (see ‘Methods’ for details). dinBplexA, a LexA repressor binding site mutant of the dinB promoter. Fluorescence intensities from single cells were measured for more than 90 × 103 cells using a flow cytometer. Means ± SDs (n > 90 × 103). The statistical significance of the difference between indicated pairs was determined by an unpaired t-test. ****, p < 0.0001. Similar results were reproduced twice.

Source data

Extended Data Fig. 5 SSB-driven ectopic localization of CLIPs.

a. Viable cell counts for +lacO250 dinB+ or ΔdinB strains (Fig. 5a) upon induction of LacIΔC11-SSB-Ct (Ct), LacIΔC11-ssb-CtDA,ΔF (CtDA,ΔF) or mock (-). Means ± SDs of triplicate measurements. Similar results were reproduced twice. b. Sensitivity of indicated assay strains to MMS upon induction of Ct or CtDA,ΔF. c. The C-terminal unstructured linker of SSB (SSBIDL in Fig. 1a) is not necessary for the formation of an ectopic SSB-Ct cluster that sensitizes cells to NFZ. Expression of LacIΔC11-SSB-Ct (Ct) or LacIΔC11-SSBIDL-Ct (IDL-Ct) comparably sensitized cells to NFZ. Addition of IPTG or binding mutations on SSB-Ct (CtDA,ΔF) eliminated this sensitization. SSBIDL-Ct, the entire C terminal unstructured linker of SSB including both SSBIDL and SSB-Ct. d. Sensitivity of the indicated strains to NFZ. Similar results were reproduced twice. e. Sensitivity of indicated assay strains to HU upon induction of Ct or CtDA,ΔF. Similar results were reproduced twice. f. ΔdinB is epistatic to polA-recQWH or hda-recQWH in sensitivity to NFZ. Sensitivity of the indicated strains to NFZ. polA- or hda-recQWH, the coding sequence for Pol I- or Hda-RecQWH. g. Sensitivity of indicated strains to NFZ. hda-recQWH or WH(R425A,R503A), the coding sequence for Hda-RecQWH or WH(R425A,R503A). h. Over-production of Crfc-RecQWH results in massive cell death. Expression of indicated genes was induced from a Tet-inducible genomic expression cassette. crfc-recQWH or WH(R425A,R503A), the coding sequence for Crfc-RecQWH or WH(R425A,R503A). Similar results were reproduced twice.

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Supplementary information

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Supplementary Figures 1–5.

Reporting Summary.

Supplementary Table 1

E. coli strains and plasmids used in this study.

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Chang, S., Thrall, E.S., Laureti, L. et al. Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis. Nat Struct Mol Biol 29, 932–941 (2022). https://doi.org/10.1038/s41594-022-00827-2

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