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The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

Abstract

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases1,2. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket2,3,4,5. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials6,7. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism5. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

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Figure 1: Crystallographic reconstruction of the reaction trajectory for 3mA excision.
Figure 2: Crystallographic snapshots of 3d3mA excision by AlkD.
Figure 3: 3mA recognition and excision through charge–dipole and CH–π interactions.
Figure 4: Excision of N3-yatakemycyladenine by AlkD.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5CL3, 5CL4, 5CL5, 5CL6, 5CL7, 5CL8, 5CL9, 5CLA, 5CLB, 5CLC, 5CLD and 5CLE.

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Acknowledgements

We thank C. Rizzo and T. Johnson Salyard for assistance with oligodeoxynucleotide synthesis and E. Skaar and L. Mike for assistance with genetic manipulation of B. anthracis. This work was funded by the National Science Foundation (MCB-1122098 and MCB-1517695 to B.F.E.) and the National Institutes of Health (R01ES019625 to B.F.E. and R01CA067985 to S.S.D.). Support for the Vanderbilt Robotic Crystallization Facility was also provided by the National Institutes of Health (S10RR026915). Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy Office of Science by Argonne National Laboratory, was supported by the US Department of Energy (DE-AC02-06CH11357). Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (085P1000817). E.A.M. and Z.D.P. were partially supported by the Vanderbilt Training Program in Environmental Toxicology (T32ES07028).

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Contributions

E.A.M. and B.F.E. conceived the project; E.A.M., R.S., Z.D.P. and B.F.E. designed experiments; E.A.M. performed biochemical and structural experiments; R.S. performed cellular experiments; Z.D.P. performed computational experiments; P.K.Y., S.S.D. and Y.I. supplied reagents; E.A.M., R.S., Z.D.P. and B.F.E. analysed data and wrote the paper; all authors commented on the manuscript.

Corresponding author

Correspondence to Brandt F. Eichman.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Remodelled DNA structures.

DNA binding by AlkD bends the helical axis by 30° and widens the minor groove by 4 Å. Remodelling of this type reduces the energetic barrier to base pair opening but does not force base flipping54,55,56. In the AlkD–3d3mA-DNA complex, this distortion creates an equilibrium between Watson–Crick and sheared conformations of the 3d3mA•T base pair. In the sheared conformation, the 3d3mA nucleobase is displaced by 4 Å into the widened minor groove but remains partially stacked in the DNA duplex. a, Sheared 3d3mA•T base pair in the AlkD–DNA complex. b, Watson–Crick 3d3mA•T base pair in the AlkD–DNA complex. c, Simulated Watson–Crick A•T base pair in the AlkD–DNA complex. Adenine was generated by removing the methyl substituent from 3d3mA and changing carbon to nitrogen at the 3-position. Computational optimization was not performed. d, Watson–Crick A•T base pair in B-form DNA (PDB accession 1BNA). The asterisks in a and b indicate the position of the methyl substituent at C3. All structures are depicted as stereo images.

Extended Data Figure 2 3d3mA substrate and product structures.

The AlkD–3d3mA-DNA complex was crystallized under moderately acidic (pH 5.7) conditions, causing a small fraction (~1%) of 3d3mA to become protonated and activated for enzymatic excision. Reaction progress was monitored by flash-freezing crystals at various times after preparing the protein–DNA complex and refining the fractional occupancies of substrate and product in the structures. a, Structure after 4 h containing only 3d3mA-DNA substrate. The 3d3mA•T base pair is present as a mixture of Watson–Crick (thin bonds) and sheared (thick bonds) conformations. b, Structure after 48 h containing a mixture of 3d3mA-DNA substrate together with AP-DNA product and 3d3mA nucleobase. c, Structure after 360 h containing only AP-DNA product and 3d3mA nucleobase. All panels show annealed omit electron density contoured to 2.5σ.

Extended Data Figure 3 Additional substrate structures.

AlkD was crystallized with two alternate 9-mer 3d3mA-DNA constructs to verify that crystal packing contacts were not significantly influencing the conformation of the DNA. As in the catalytic 12-mer substrate complex, 3d3mA remained stacked in the duplex in both 9-mer structures. However, unlike in the 12-mer complex, a mixture of Watson–Crick and sheared conformations was not observed. Instead, the 3d3mA•T base pair in each construct is either entirely in the Watson–Crick conformation or entirely in the sheared conformation. Formation of the AP product was not observed in either 9-mer construct, probably as a consequence of the neutral (pH 7.0) crystallization conditions, under which the fraction (<0.1%) of 3d3mA activated for depurination would be drastically reduced. a, Watson–Crick conformation. b, Sheared conformation. Both panels show annealed omit electron density contoured to 2.5σ.

Extended Data Figure 4 Comparison of catalytic and non-catalytic AlkD–3d3mA-DNA complexes.

AlkD binds 3d3mA-DNA in two orientations that differ only in the position of the 3d3mA•T base pair about its dyad axis. Either 3d3mA (catalytic complex) or the opposing thymine (non-catalytic complex) resides against the protein surface. Both orientations use a common set of interactions that induce bending of the helical axis and widening of the minor groove, causing shearing of the 3d3mA•T base pair. In either complex, the nucleotide adjacent to the protein surface shows the same degree of displacement into the minor groove. The asymmetric position of the 3d3mA nucleotide in the duplex allows crystal packing interactions to select for a single orientation from a likely mixture of orientations in solution. Different DNA constructs were used to exploit these packing interactions and crystallize complexes in each binding orientation separately. Positive charge on the deoxyribose of 3mA would produce stronger electrostatic interactions with Trp109, Asp113 and Trp187 that would likely favour the catalytic binding orientation as well as a sheared conformation of a 3mA•T base pair. a, Catalytic orientation. The Watson–Crick conformer of the 3d3mA•T base pair was omitted for clarity. b, Non-catalytic orientation (PDB accession 3JX7).

Extended Data Figure 5 Electrostatic potential surfaces and computed binding energies.

Charge transfer from the catalytic ensemble (Trp109, Asp113, Trp187 and Asp113-associated water) to the modified nucleosides stabilizes the AlkD–DNA complexes. a, 3d3mA substrate. b, 3mA substrate. c, Transition state (TS) approximation. d, dR+ intermediate. Positive charge located on the deoxyribose moiety of the unbound nucleosides relative to that on the unbound dR+ intermediate is indicated below the lesions. Charge transferred from the catalytic ensemble to the lesions upon complex formation is indicated above the arrows. Electrostatic potentials were scaled to −0.22–0.55 atomic units on an isodensity surface of 0.05 electrons bohr−3. e, Modified nucleosides shown in ad. f, Computed binding energies (kcal mol−1), differential bond elongation energies (ΔΔE, kcal mol−1), and corresponding rate enhancements (r.e.).

Extended Data Figure 6 Growth curves and spot assays.

Wild-type (black) and alkD-knockout (red) strains of B. anthracis were treated with varying concentrations of mMS and YTM. Control experiments contained no drug. Deletion of AlkD caused no observable phenotype with mMS but resulted in increased sensitivity to YTM. a, mMS treatment. b, YTM treatment. Error bars represent the s.e.m. from three replicate growth curves. Spot assays were performed in duplicate.

Extended Data Table 1 X-ray data collection and refinement statistics
Extended Data Table 2 X-ray data collection and refinement statistics
Extended Data Table 3 X-ray data collection and refinement statistics

Supplementary information

Supplementary Data

This file contains atomic coordinates for computationally optimised structures. (XLSX 36 kb)

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Mullins, E., Shi, R., Parsons, Z. et al. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions. Nature 527, 254–258 (2015). https://doi.org/10.1038/nature15728

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