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. 2009 Jun 11;459(7248):808-13.
doi: 10.1038/nature08076.

Flipping of alkylated DNA damage bridges base and nucleotide excision repair

Julie L Tubbs  1 , Vitaly Latypov, Sreenivas Kanugula, Amna Butt, Manana Melikishvili, Rolf Kraehenbuehl, Oliver Fleck, Andrew Marriott, Amanda J Watson, Barbara Verbeek, Gail McGown, Mary Thorncroft, Mauro F Santibanez-Koref, Christopher Millington, Andrew S Arvai, Matthew D Kroeger, Lisa A Peterson, David M Williams, Michael G Fried, Geoffrey P Margison, Anthony E Pegg, John A Tainer
Affiliations

Flipping of alkylated DNA damage bridges base and nucleotide excision repair

Julie L Tubbs et al. Nature. .

Abstract

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O(6)-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O(6)-methylguanine or cigarette-smoke-derived O(6)-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

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Figures

Figure 1
Figure 1. Atl1 structure and lesion-binding site
a, Overlay of Atl1 (yellow) and AGT (cyan; pdb 1EH6) models and comparison of key functional residues. 2Fo-Fc electron density (blue) for Atl1 with the binding site Trp56 side chain omitted. b, Atl1 (magenta) bound to DNA containing O6-mG (orange). 2Fo-Fc simulated annealing composite omit map (blue) shown for DNA. c and d, Atl1 lesion-binding site close-up with O6-pobG (c) or O6-mG (d). Amino acid side chains (ball-and-stick) and hydrogen bonds to the damaged guanine (green dashes) show the damage binding.
Figure 2
Figure 2. Atl1 DNA binding and damage sculpting
a, Overlay of DNA-free (yellow) and DNA-bound (magenta) Atl1 with the hAGT C-terminal domain (cyan). The rotated O6-mG (center, spheres) is shown with the binding site loop that determines the open or closed conformation of Atl1. b, Atl1 molecular surface revealing an “open” state. c, DNA-bound Atl1 molecular surface showing the protein “closed” state. d, Atl1-DNA interaction schematic.
Figure 3
Figure 3. Atl1 DNA lesion binding affinity and stoichiometry
a, Atl1 binding and dissociation for oligonucleotides containing O6-mG (top), O6-pobG (center), or abasic site (bottom). b, Gel-shift assays for Atl1 binding normal and O6-mG 13mer dsDNA (left) and associated O6-mG DNA binding isotherm analysis (right) showing two independent experiments (●,■). c, Sedimentation equilibrium data for O6-mG 13mer dsDNA complexes (left) and normal 26mer dsDNA (right). Small, randomly distributed residuals (top panels) indicate models in which free protein, DNA, and one protein-DNA complex equilibrate in solution. Calculated stoichiometries are 1.15 ± 0.08 for O6-mG ds13mer and 3.03 ± 0.20 for normal ds26mer.
Figure 4
Figure 4. Biochemical and genetic connection of Atl1 to NER
a–b, Coomassie-stained gel (left) and far-western blot (right) probed with FLAG-eAtl (a) or FLAG-UvrA (b). c, MNNG-induced (top) and spontaneous (bottom) mutations of wild-type, Δatl1, Δrad13 and Δatl1 Δrad13 S. pombe strains. d, Atl1 and Rad13 are epistatic for MNNG toxicity. Serial dilutions of wild-type, Δatl1, Δrad13 and Δatl1 Δrad13 S. pombe cells spotted on yeast extract (YE) plates or YE plates containing 0.08 µg/ml MNNG. Results shown are mean ± s.d.; n ≥ 3. e–h, Clonogenic assay, revealing Atl1 is epistatic for MNNG toxicity with Rad13 (e) and Swi10 (f), but not Rhp14 (g) or Rad2 (h). Results shown are mean ± s.e.m; n ≥ 3.
Figure 5
Figure 5. Alkyl-G lesion recognition allows NER repair of relatively non-distorting base lesions
The distorted, stable ATL-DNA complex creates a platform to recruit NER enzymes to O6alkyl-G lesions. This general model is based upon our combined structural, biochemical and genetic results.
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