Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation-π interactions

Abstract

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O(6)-alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O(6)-alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O(6)-alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O(6)-alkylguanine, as determined using molecular mechanics calculations. An unexpected consequence of this feature is the recognition of 2,6-diaminopurine and 2-aminopurine, as confirmed in crystal structures of respective Atl1-DNA complexes. O(6)-Alkylguanine and guanine discrimination is diminished for Atl1 R69A and R69F mutants, and S. pombe R69A and R69F mutants are more sensitive toward alkylating agent toxicity, revealing the key role of Arg69 in identifying O(6)-alkylguanines critical for NER recognition.

Fig. 1.

Structures of guanine, modified purines, and ODNs used in study. (A) Guanine (numbered), O⁶-alkylguanine, and modified purines incorporated into 13-mer ODN substrate (B) (X = guanine, O⁶-alkylguanine, or modified purine).

Fig. 2.

Analysis of binding of ATL proteins to DNA monitored using fluorescence emission intensity. Atl1 (A) or MBP-TTHA1564 (B) were added to 1 nM ODN 5′-SIMA(HEX)-GCCATGXCTAGTA (X = O⁶-carboxymethylguanine) in buffer (50 mM Tris.HCl (pH 7.5), 50 mM NaCl, 1mM EDTA) and fluorescence emission measured at 560 nm with excitation at 530 nm.

Fig. 3.

Equilibrium dissociation constants (K_D values) determined for ATL–DNA complexes using ss ODNs containing O⁶-alkylguanine and other purine bases (structures, analog abbreviations, and the ODN sequence are shown in Fig. 1) with the following proteins: (A) Atl1 from S. pombe, (B) MBP-TTHA1564 from T. thermophilus, (C) Atl1 wild type (red), Atl1 mutant proteins R69F (blue) and R69A (yellow), (D) Atl1 wild type, Atl1 R69F and R69A mutants with ss ODNs containing O⁶-alkylguanine (mean K_D values) (gold) or guanine (green). Log K_D values are displayed in C and D.

Fig. 4.

Calculated MEP surfaces for guanine, O⁶-methylguanine, and other purines. Black circles highlight the regions on the purine bases that contact Arg69 (interatomic separations less than 3.5 Å). Colors correspond to: blue +50 kJ/mol, green neutral, red −50 kJ/mol. The site to which the anomeric carbon (C1') of the nucleoside is attached to the purine is indicated in each figure.

Fig. 5.

Crystal structures of (A) Atl1:O⁶-MeG-DNA duplex, (B) Atl1:DAP-DNA duplex, and (C) Atl1:2-AP-DNA duplex ODN complexes. 2F_o-F_c (1 σ) and F_o-F_c (+3 σ) electron density maps with DAP (B) or 2-AP (C) omitted from the model are shown in blue and cyan, respectively.

Fig. 6.

Growth inhibition assays with S. pombe and MNNG. (A) Analysis of atl1 expression by SDS/PAGE of whole-cell extracts followed by Western blot with anti-Atl1 antibody. Purified recombinant Atl1 protein was used as a positive control. (B) Loading control for the Western blot analysis. Equal amounts of proteins (15 µg per lane) were loaded onto two 4–12% NuPAGE Bis-Tris Pre-Cast Gels. One gel was probed with anti-Atl1 antibody, whereas a second gel was stained in 0.05% (wt/vol) Coomassie Brilliant Blue R250. Wild type or atl⁺–wild type (GM1); atl1Δ or Δ - full deletant of the atl1 gene (GM3); atl1-R69A or 69A (GM177); atl1-R69F or 69F (GM178). M, BenchMark Pre-Stained Protein Ladder (Invitrogen). (C) Agar plate (“spot”) assay for atl1-R69A and atl1-R69F mutants after treatment with MNNG. Aliquots of 10-fold serial dilutions were spotted on plates with YEA media (growth medium containing yeast extract, glucose, and adenine sulfate) containing MNNG under indicated concentrations and photographed after 5 d.