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The functional coupling between restriction and DNA phosphorothioate modification systems underlying the DndFGH restriction complex


Bacteria have evolved diverse mechanisms to cope with the constant threat of phage predation. DndABCDE-catalysed DNA phosphorothioate modification, in which the non-bridging oxygen of the DNA sugar-phosphate backbone is replaced by sulfur, can couple with proteins encoded by the dndFGH gene cluster to provide defensive protection, albeit via an obscure mode of action. Here we present structural and biochemical insights showing that the proteins DndF, DndG and DndH form a DndFGH complex and exhibit functions that are not detected in the individual proteins, including DNA nicking and translocation activities. Interestingly, DndFGH shows preferential affinity for phosphorothioate DNA, which suppresses the DNA-stimulated ATPase activity of the complex and consequently impedes its translocation and nicking activities. This phosphorothioate binding preference is believed to account for self/non-self discrimination and enable DndFGH to specifically introduce nicking damage in invasive DNA. This study expands our knowledge of the defence mechanisms involving epigenetic markers for the protection of self-DNA.

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Fig. 1: DndFGH provides protection against invasive parasites.
Fig. 2: DNA nicking assays of the DndFGH complex in vitro.
Fig. 3: ATPase activity of DndF and its involvement in DndFGH nicking.
Fig. 4: Structural and biochemical analyses of DndG from E. coli B7A.
Fig. 5: Structural and biochemical analyses of DndH.
Fig. 6: Effect of the translocation activity of DndH on DndFGH defence.
Fig. 7: Effects of DNA and ATP on the conformational changes of E.DndFGH.
Fig. 8: Regulation of E.DndFGH activities by DNA PT modification.

Data availability

The data supporting the findings of this study are available from the corresponding authors upon reasonable request. The coordinates and structure factors of DndG and DndHCTD from E. coli B7A have been deposited in the Protein Data Bank under the accession numbers 7EXX and 7ES4, respectively. Source data are provided with this paper.


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We thank the staff of the BL19U1 beamline of the National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility for assistance in data collection. This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31925002 to Shi Chen, 32125001 to L.W., and 31872627 and 32170030 to G.W.), the National Key Research and Development Program of China (grant nos. 2022YFA0912200 to L.W., 2022YFA0912500 to Shi Chen. and 2020YFA0907300 to G.W.) and the Shanghai Jiao Tong University Scientific and Technological Innovation Fund (to G.W.).

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Authors and Affiliations



Shi Chen, G.W. and L.W. conceived the study and supervised the project. D.W., Y.T., Siwei Chen, Y.H. and X.C. performed the experiments. D.W., Y.T., Siwei Chen, Y.H., X.C., W.Z., Z.D., Z.L., L.W., G.W. and Shi Chen analysed the data. D.W., L.W., G.W. and Shi Chen wrote the manuscript.

Corresponding authors

Correspondence to Lianrong Wang, Geng Wu or Shi Chen.

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The authors declare no competing interests.

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Nature Catalysis thanks Weixin Tang, Konstantin Severinov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 SDS–PAGE gel and SEC-MALS analysis of the E.DndFGH complex.

a, Gel filtration profile of His-DndF, His-DndG, His-DndH and the DndFGH complex of B7A. The E.DndFGH complex was prepared by mixing His-DndF (64 μM), His-DndG (64 μM) and His-DndH (32 μM). b, A gel image of the SDS–PAGE analysis of eluted protein fractions from the size-exclusion chromatography. Results are representative of three independent experiments. c, No obvious DNA was observed in the eluted protein fractions by agarose gel electrophoresis. To exclude the involvement of DNA in the formation of the DndFGH complex, the mixture of DndF, DndG and DndH was treated with DNase I (0.033 U/µl) at 37 °C for 10 min. DNase I is capable of digesting DNA up to 5 μg under our experimental conditions. Results are representative of three independent experiments. d, SEC-MALS analysis shows that the E.DndFGH complex elutes with a molecular mass of 434.03 kDa, consistent with a theoretical value of 433.44 kDa when DndF, DndG and DndH, as shown in (a), assemble into a complex in a molar ratio of 2:2:1. The left and right axes represent the molecular weight and the light scattering detector reading, respectively. The black curve represents the calculated molecular weight. Results are representative of three independent experiments.

Source data

Extended Data Fig. 2 Nonspecific nicking activity of E.DndFGH.

Run-off sequencing of open circular pUC19 treated with DndFGH or Nt. BspQI. The peaks in the sequencing chromatogram dropped dramatically at the sequence-specific nicking site of Nt.BspQI. In contrast, no such sequencing peak drop was observed when DndFGH-nicked pUC19 DNA was used as a template despite the presence of the 5′-GAAC-3′/5′-GTTC-3′ motif (red underlined).

Extended Data Fig. 3 Structural comparison.

Structural superimposition showing the similarities between the DndHCTD structure (hot pink, PDB code: 7ES4) and VirB4CTD (cyan, PDB code: 4AG5), HerA (green/cyan, PDB code: 4D2I) and TrwBΔN70 (slate, PDB code: 1E9S).

Extended Data Fig. 4 Translocation assays.

a–c, Streptavidin displacement over time from biotinylated DNA (250 ng) ranging from 150 to 750 bp is shown in the presence of E.DndFGH (a), E.DndFGHK1354A (b) or E.DndFGHD1574A (c). The molar ratio between E.DndFGH and DNA is approx. 5:1. d, The stacked column chart shows the relative percentages of free DNA and streptavidin-bound DNA (SA DNA). The results were obtained by quantitation of the gels in panel (a). Values represent the mean ± SD of three independent streptavidin displacement experiments. e, The steady-state rate of ATPase hydrolysis as a function of the DNA length and DNA concentration. Concentration is plotted as micromolar-base pairs (μMbp). Values represent the mean ± SD from three biologically independent experiments. f, g, The trends are fit with the Michaelis–Menten equation, allowing for the extraction of the Km (f) and Vmax (g) parameters. Data represent the mean ± SD from three biologically independent experiments.

Source data

Extended Data Fig. 5 A schematic model of PT modification-modulated DndFGH defence.

a, DndA/IscS and DndBCDE proteins act as the modification component to confer PT modification at a subset of 5′-GAAC-3′/5′-GTTC-3′ sites in host DNA, generating 5′-GPSAAC-3′/5′-GPSTTC-3′. b, As the restriction component, the DndFGH complex undergoes conformational changes to generate DNA nicking and translocation activities. Furthermore, PTs in self-DNA suppress the DNA-stimulated ATP hydrolysis activity of the DndFGH complex and consequently impedes its translocation and nicking activities, thus preventing autoimmunity.

Extended Data Fig. 6 Self-vs-nonself discrimination of DndFGH in vitro.

When PT-modified (PT+) pUC19 and unmodified (PT) pACYC184 DNA were mixed at a 1:1 ratio and subjected to the nuclease digestion of E.DndFGH, PT+ pUC19 remained intact, whereas pACYC184 was specifically damaged. Without PT modification, PT pUC19 DNA is also susceptible to E.DndFGH. Results are representative of three independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Figs. 1–3.

Reporting Summary

Supplementary Data

Supplementary Tables 3–5.

Source data

Source Data Fig. 1

Unprocessed plates, statistical source data.

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Source Data Fig. 5

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Unprocessed gels, statistical source data.

Source Data Extended Data Fig. 1

Unprocessed gels, statistical source data.

Source Data Extended Data Fig. 4

Unprocessed gels, statistical source data.

Source Data Extended Data Fig. 6

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Wu, D., Tang, Y., Chen, S. et al. The functional coupling between restriction and DNA phosphorothioate modification systems underlying the DndFGH restriction complex. Nat Catal 5, 1131–1144 (2022).

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