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.
This is a preview of subscription content, access via your institution
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Maynard, S., Schurman, S. H., Harboe, C., de Souza-Pinto, N. C. & Bohr, V. A. Base excision repair of oxidative DNA damage and association with cancer and aging. Carcinogenesis 30, 2–10 (2008).
Nabel, C. S., Manning, S. A. & Kohli, R. M. The curious chemical biology of cytosine: deamination, methylation, and oxidation as modulators of genomic potential. ACS Chem. Biol. 7, 20–30 (2012).
Iyer, L. M., Zhang, D., Maxwell Burroughs, A. & Aravind, L. Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA. Nucleic Acids Res. 41, 7635–7655 (2013).
Mohni, K. N. et al. HMCES maintains genome integrity by shielding abasic sites in single-strand DNA. Cell 176, 144–153.e13 (2019).
Aravind, L., Anand, S. & Iyer, L. M. Novel autoproteolytic and DNA-damage sensing components in the bacterial SOS response and oxidized methylcytosine-induced eukaryotic DNA demethylation systems. Biol. Direct 8, 20 (2013).
Srivastava, M. et al. Replisome dynamics and their functional relevance upon DNA damage through the PCNA interactome. Cell Rep. 25, 3869–3883.e4 (2018).
Lindahl, T. DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli. J. Biol. Chem. 252, 3286–3294 (1977).
Hirel, P. H., Schmitter, M. J., Dessen, P., Fayat, G. & Blanquet, S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl Acad. Sci. USA 86, 8247–8251 (1989).
Roberts, J. C., Charyulu, R. L., Zera, R. T. & Nagasawa, H. T. Protection against acetaminophen hepatotoxicity by ribose-cysteine (RibCys). Pharmacol. Toxicol. 70, 281–285 (1992).
Joint Commission on Biochemical Nomenclature Abbreviations and symbols for the description of conformations of polynucleotide chains. Pure Appl. Chem. 55, 1273–1280 (1983).
Liu, M. et al. The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo. Proc. Natl Acad. Sci. USA 107, 4925–4930 (2010).
Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).
Landau, M. et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299–W302 (2005).
Jurrus, E. et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 27, 112–128 (2018).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013).
Winn, M. D. et al. Overview of the CCP 4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Rychlewski, L., Li, W., Jaroszewski, L. & Godzik, A. Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Sci. 9, 232–241 (2008).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Steiner, R. A., Lebedev, A. A. & Murshudov, G. N. Fisher’s information in maximum-likelihood macromolecular crystallographic refinement. Acta Crystallogr. D 59, 2114–2124 (2003).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Chalk, R. in Heterologous Gene Expression in E. coli Vol. 1586 (ed. Burgess-Brown, N. A.) 373–395 (Humana Press, 2017).
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.
The authors declare no competing interests.
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
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.
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).
About this article
Cite this article
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
This article is cited by
HMCES safeguards genome integrity and long-term self-renewal of hematopoietic stem cells during stress responses
Nature Structural & Molecular Biology (2022)
Nature Structural & Molecular Biology (2019)