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Protection of abasic sites during DNA replication by a stable thiazolidine protein-DNA cross-link

Nature Structural & Molecular Biology volume 26, pages 613–618 (2019)Cite this article

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Abstract

Abasic (AP) sites are one of the most common DNA lesions that block replicative polymerases. 5-hydroxymethylcytosine binding, embryonic stem cell-specific protein (HMCES) recognizes and processes these lesions in the context of single-stranded DNA (ssDNA). A HMCES DNA-protein cross-link (DPC) intermediate is thought to shield the AP site from endonucleases and error-prone polymerases. The highly evolutionarily conserved SOS-response associated peptidase (SRAP) domain of HMCES and its Escherichia coli ortholog YedK mediate lesion recognition. Here we uncover the basis of AP site protection by SRAP domains from a crystal structure of the YedK DPC. YedK forms a stable thiazolidine linkage between a ring-opened AP site and the α-amino and sulfhydryl substituents of its amino-terminal cysteine residue. The thiazolidine linkage explains the remarkable stability of the HMCES DPC, its resistance to strand cleavage and the proteolysis requirement for resolution. Furthermore, its structure reveals that HMCES has specificity for AP sites in ssDNA at junctions found when replicative polymerases encounter the AP lesion.

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Fig. 1: Stability analysis of the human HMCES SRAP-abasic site DNA-protein cross-link.
Fig. 2: YedK DPC crystal structure.
Fig. 3: The SRAP DPC forms a thiazolidine linkage stabilized by conserved residues.
Fig. 4: SRAP can accommodate dsDNA 3′ to the AP site.

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Data availability

Structures have been deposited in the Protein Data Bank under accession codes 6NUA (DPC) and 6NUH (C3-spacer). Source data for Figs. 1b,c, 3d, 4d,e and Supplementary Fig. 4 are available with the paper online as Source Data for Figs. 1, 3, and 4. All other data are available upon request.

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Acknowledgements

This work was supported by NIH grants nos. R01ES030575 (D.C.), R01GM117299 (B.F.E.) and P01CA092584 (D.C. and B.F.E.). P.S.T. was supported by F30CA228242, K.M.A. was supported by the Vanderbilt Molecular Biophysics Training Program (T32GM08320) and K.N.M. was supported by T32CA009582. Core facilities were supported by the Vanderbilt-Ingram Cancer Center P30CA068485. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Use of LS-CAT Sector 21 beamline was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817).

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Author notes

  1. These authors contributed equally: Petria S. Thompson, Katherine M. Amidon.

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA

    Petria S. Thompson, Kareem N. Mohni, David Cortez & Brandt F. Eichman

  2. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Katherine M. Amidon & Brandt F. Eichman

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  1. Petria S. Thompson
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Contributions

P.S.T., K.M.A. and K.N.M designed and completed the experiments, interpreted results and edited the manuscript. D.C. and B.F.E. supervised and funded the project, designed experiments and wrote the manuscript.

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Correspondence to David Cortez or Brandt F. Eichman.

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The authors declare no competing interests.

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Peer review information: Beth Moorefield 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 SRAP conservation.

a. Structure-based sequence alignment between E. coli YedK and human HMCES SRAP domain, together with a sequence alignment of SRAP domains from 8 additional species. Secondary structure from the YedK DPC structure is shown above the alignment. Symbols above specific residues denote those involved in binding DNA (circles), distorting DNA at the 5′ side of the AP site (wedge, blue circles), stacking against dsDNA immediately 3′ to the AP site (shelf, magenta circles), and stabilizing the thiazolidine crosslink (Cys2 pocket, “×”). b. Orthogonal views of the YedK DPC structure colored rainbow from N- (blue) to C-terminus (red). c. Superposition of E. coli YedK DPC (blue/gold) and human HMCES SRAP domain (PDB ID 5KO9, silver). The r.m.s.d. between the structures is 1.40 Å for all backbone atoms. d. Superposition of YedK DPC (blue/magenta) and free YedK (orange). Loops in YedK DPC that are disordered in the free protein are colored magenta. Proteins are shows as a Cα-backbone trace.

Supplementary Figure 2 Details of the SRAP active site.

Stereo views of residues contacting the thiazolidine linkage in the two protein-DNA complexes in the asymmetric unit of the YedK DPC structure, superimposed with 1σ 2Fo-Fc electron density. Dashed lines and numbers indicate lengths of hydrogen bonds in Å.

Supplementary Figure 3 Structural details of the non-covalent SRAP-DNA complex.

a. Superposition of YedK covalently crosslinked to AP-DNA (blue) and non-covalently bound to C3-spacer DNA (magenta). The abasic site is marked with an asterisk. The double-headed arrow shows the most significant difference between the two structures—movement of β-hairpin (β7- β8) that stabilizes the backbone of the DNA 3′ to the AP site. b. Stereo view of the YedK active site in the YedK/C3-spacer-DNA structure, superimposed with 1σ 2Fo-Fc electron density. The C3-spacer is colored green and flanking nucleotides gold. Dashed lines and numbers indicate lengths of hydrogen bonds in Å. c. DNA in the DPC (top) and non-covalent C3-spacer (bottom) structures, colored by B-factor. d. Average B-factor of each nucleotide in the DPC (black) and non-covalent C3-spacer (blue) structures.

Supplementary Figure 4 Binding of HMCES SRAP domain to ssDNA and ssDNA/dsDNA junctions containing a tetrahydrofuran (THF) abasic site analog.

Binding was monitored by a change in fluorescence anisotropy as protein was titrated against DNAs that contained a FAM label at the 5′-end of the THF strand. The maximal change in anisotropy (amplitude of binding isotherm at saturation) is dependent on the tumbling rate of the FAM label, which is different for each of the three substrate. Dissociation constants (Kd) were derived from non-linear least squares fit of a two-state, single-site binding model to the data.

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Thompson, P.S., Amidon, K.M., Mohni, K.N. et al. Protection of abasic sites during DNA replication by a stable thiazolidine protein-DNA cross-link. Nat Struct Mol Biol 26, 613–618 (2019). https://doi.org/10.1038/s41594-019-0255-5

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