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Structural basis of HMCES interactions with abasic DNA and multivalent substrate recognition

Abstract

Embryonic stem cell-specific 5-hydroxymethylcytosine-binding protein (HMCES) can covalently cross-link to abasic sites in single-stranded DNA at stalled replication forks to prevent genome instability. Here, we report crystal structures of the human HMCES SOS response-associated peptidase (SRAP) domain in complex with DNA-damage substrates, including HMCES cross-linked with an abasic site within a 3′ overhang DNA. HMCES interacts with both single-strand and duplex segments of DNA, with two independent duplex DNA interaction sites identified in the SRAP domain. The HMCES DNA-protein cross-link structure provides structural insights into a novel thiazolidine covalent interaction between the DNA abasic site and conserved Cys 2 of HMCES. Collectively, our structures demonstrate the capacity for the SRAP domain to interact with a variety of single-strand- and double-strand-containing DNA structures found in DNA-damage sites, including 5′ and 3′ overhang DNAs and gapped DNAs with short single-strand segments.

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Fig. 1: Interactions between SRAPd and 3′ overhang DNA.
Fig. 2: SRAPd interaction with potential DNA-damage repair substrates.
Fig. 3: Crystal structure of the human HMCES SRAPd cross-linked to a DNA abasic site.

Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes: 5KO9 for Apo_SRAPd, 6OEB for SRAPd_3nt, 6OEA for SRAPd_6nt, 6OE7 for SRAPd_DPC. LC–MS data underlying Supplementary Fig. 3 have been deposited in Zenodo (https://doi.org/10.5281/zenodo.2662532). Source data for Fig. 2d and Supplementary Fig. 1 are available with the paper online.

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Acknowledgements

We are grateful to H. Wyatt for fruitful discussions, advice from P.J. Brown and W. Tempel on interpretation of DNA-protein cross-link chemistry and S. Ackloo on mass spectrometry data analysis. Special thanks to S. Duan for preparing the DNA abasic site digestion. We also thank U. Chinte, J. Chrzas, N. Duke and Z. Jin from SERCAT 22ID-D beamline for collecting the initial SRAPd_DPC datasets. This research used resources of the Advanced Light Source, which is a Department of Energy Office of Science User Facility under contract no. DE-AC02-05CH11231. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center (SBC) at the Advanced Photon Source. SBC-CAT is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract no. DE-AC02-06CH11357. This work is based on research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (no. P41 GM103403). The Pilatus 6M detector on beamline 24-ID-C is funded by a NIH Office of Research Infrastructure Programs High End Instrumentation grant (no. S10 RR029205). The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie; Bayer Pharma AG; Boehringer Ingelheim; Canada Foundation for Innovation; Eshelman Institute for Innovation; Genome Canada through Ontario Genomics Institute (no. OGI-055); Innovative Medicines Initiative (EU/EFPIA) (no. ULTRA-DD: 115766); Janssen, Merck & Co.; Novartis Pharma AG; Ontario Ministry of Research Innovation and Science; Pfizer, São Paulo Research Foundation-FAPESP, Takeda and the Wellcome Trust. This research is also supported by the Canadian Institutes of Health Research (no. FDN154328) and Natural Sciences and Engineering Research Council (no. RGPIN-2015-05939) to C.H.A., intramural funds of the National Library of Medicine, NIH, to L.A. and the National Cancer Institute (no. R35 CA210043) to A.R.

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Authors

Contributions

L.H. performed the experiments. Y.L., M.R. and H.Z. cloned, expressed and purified the proteins. A.R. and C.H.A. conceived the project. L.H., L.A., A.R. and C.H.A. contributed to experimental design and review of data. L.H. and C.H.A. wrote the paper.

Corresponding author

Correspondence to Cheryl H Arrowsmith.

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The authors declare no competing interests.

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Peer review information: Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Fluorescence polarization DNA-binding assays for HMCES SRAP domain.

Comparison of DNA-binding activities of wild-type (W.T.) and mutant SRAPd variants by fluorescence polarization, using single-stranded DNA (upper panel) and dsDNA containing a 3-nucleotide gap (3-nt gap DNA) (lower panel). Experiments were performed in triplicates and data are represented as mean ± s.d. NB, no detectable binding.Source data.

Source data

Supplementary Figure 2 Close-up view of the ssDNA binding cleft of the HMCES SRAP domain.

The mFo-DFc electron density omit-maps for the ssDNA segment of 3′ overhang DNA in SRAPd_3nt (left panel), and SRAPd_6nt (right panel), displayed as magenta mesh and contoured at 2.5σ.

Supplementary Figure 3 Raw and deconvoluted mass spectra for the HMCES SRAP domain crosslinked to DNA abasic site.

(a) Raw and (b) deconvoluted mass spectra for the SRAP domain alone; (c) Raw and (d) deconvoluted mass spectra for the SRAP domain incubated with abasic site containing DNA (AP9 DNA).

Supplementary information

Supplementary Information

Supplementary Figs. 1-3, Supplementary Tables 1 and 2

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Source data

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Halabelian, L., Ravichandran, M., Li, Y. et al. Structural basis of HMCES interactions with abasic DNA and multivalent substrate recognition. Nat Struct Mol Biol 26, 607–612 (2019). https://doi.org/10.1038/s41594-019-0246-6

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