Fig. 4.

eATL prevents binding of MutS to O⁶-alkylguanine:T mispairs. The incubation buffer for MutS binding assays contains cold competitor DNA (1 kb ladder; New England Biolabs) at a concentration of 10 ng/mL to minimize unspecific binding of MutS to double-stranded DNA. The DNA probes are ³²P-radiolabeled oligoduplexes containing G, mG, heG, 1hpG, and 2hpG, paired to C (:C) or to T (:T). Oligonucleotides were first incubated with eATL at 0 nM (lanes 2 and 8), 125 nM (lanes 3 and 9), 250 nM (lanes 4 and 10), and 500 nM (lanes 5, 6, 11, and 12). Reactions were next supplemented with MutS at a final concentration of 1 μM (lanes 2–5 and 8–11). eATL interferes with MutS binding for all adducts except mG. The same results are observed when the order of addition of eATL and MutS is reversed.