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

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.

Fig. 1.. Stability analysis of the human HMCES SRAP-abasic site DNA protein crosslink.

a, HMCES SRAP DPC stability measured at the indicated temperatures. Free and DNA-crosslinked HMCES was detected by coomassie blue staining. The HMCES-DPC percentage in this experiment is approximately 50% because uncrosslinked DNA was removed by dialysis after a short reaction time. b, Boiling the HMCES DPC causes hydrolysis (mean ± S.D., n=3 independent measurements) c, HMCES DPC stability measured before or after denaturation by boiling for two minutes. d, HMCES SRAP domain was incubated with a 20-mer AP-site containing oligonucleotide to form a crosslink, digested with proteinase K followed by heat inactivation of the protease, and then incubated at 37°C for the times indicated. Electrophoresis and autoradiography was used to visualize the DNA. e, HMCES SRAP was incubated with 31-mer AP-DNA and digested with proteinase K, and the peptide DPC incubated with APE1 for 2 hours. Bands were visualized by Cy5 fluorescence. Uncropped gel images are shown in Supplementary Data Set 1. Source data for b,c are available online.

Fig. 2.. YedK DPC crystal structure.

a, DNA fit to 2Fo-Fc composite annealed omit electron density contoured at 1σ. b, Orthogonal views of E. coli YedK (blue) crosslinked to AP-DNA (gold). c,d, YedK solvent-accessible surface colored by electrostatic potential from −5 to +5 k_BT/e_C (c) and sequence conservation from 158 unique SRAP orthologs (d). e, Schematic of protein-DNA interactions.

Fig. 3.. The SRAP DPC forms a thiazolidine linkage stabilized by conserved residues.

a-h, a, The DPC between the AP site (green) and Cys2 (blue) superimposed against 2Fo-Fc composite annealed omit electron density contoured at 1σ. b, Proposed chemical mechanism of the crosslinking reaction with competing lyase reactions in red. c, Representative denaturing PAGE gel showing crosslinking and lyase activity of YedK mutants. Bands were visualized by FAM fluorescence. d, Crosslinking efficiencies of YedK mutants (mean ± SD, n=3 independent measurements). e, NaBH₃CN was added to crosslinking reactions to trap the Schiff base intermediates of YedK C2A and C2S mutants. The NaBH₃CN-reduced Schiff base is refractory to β-elimination. Bands were visualized by FAM fluorescence. f, Residues contacting the DPC (DNA, gold; AP site green; protein, blue). The alternate Glu105 conformer is cyan. Dashed lines denote hydrogen bonds. g, Orthogonal view showing hydrophobic residues cradling Cys2. The second Glu105 conformer is not shown for clarity. Uncropped gel images are shown in Supplementary Data Set 1. Source data for d are available online.

Fig. 4.. SRAP can accommodate dsDNA 3′ to the AP site.

a, Model of YedK DPC with a 3′ junction at AP site. The modeled complementary DNA strand is pink. The wedge domain blocking dsDNA access 5′ to the AP site is blue. b, Wedge-DNA interactions 5′ to the AP site. c, Sequence conservation of the DNA shelf that presumably stabilizes dsDNA 3′ to the AP site. d, EMSA showing binding of human HMCES SRAP domain to the indicated DNA ligands. The plot shows mean ± S.E.M from n = 3 independent measurements. Uncropped gel image is shown in Supplementary Data Set 1. e, Percent of the indicated DNA substrates crosslinked to human HMCES SRAP domain (mean ± S.D., n=3 independent measurements). Source data for d,e are available online.