Figure 4. Qualitative assessment of the binding of Atl1 to ODN by EMSA.

(A) Atl1 binds specifically to ODNs containing O⁶-alkylguanines.

ODNs were annealed by mixing complement (5′-[HEX]-GGGCCAGCACGGAGCTGCAGTTC) with the “lesion”-containing ODN (5′-GAACTXCAGCTCCGTGCTGGCCC, where X is guanine (G) or the modified bases: O⁶-methyl (Me), -carboxymethyl (CM), -ethyl (Et), -hydroxyethyl (HOEt), -n-propyl (Pr), -butyl (Bu) -benzyl (Bn), -pyridyloxobutyl (Pob) and -4-bromothenyl (BT). Atl1 (5 μM) was incubated with duplex ODN (1 μM) in total volume of 10 μL of reaction buffer then electrophoresed. Gels were scanned using a Pharos laser scanner (BioRad).

(B) Additional EMSA controls for O⁶-MeG-containing ODN which include incubation with the non-DNA binding protein MBP and incubation of Atl1, MBP or the ODN incubation mixes with proteinase K (PK). The upper gel photograph was obtained a Pharos laser scanner and the gel was electroblotted to nitrocellulose. The Atl1 (centre gel photograph) and MBP proteins (lower gel photograph) and their degradation by PK were visualized by western blot with rabbit anti-Atl1 polyclonal antiserum or anti-MBP antibodies followed by incubation with horseradish peroxidase-labeled anti-rabbit antibodies and detection by enhanced chemiluminescence. Data for all other ODNs are shown in Figure S4.