Figure 1.

Overview of the Sec-specific genetic selection system. (A) O⁶-methylguanosine within DNA and human AGT with the active site residue at position 145 indicated as ‘X’. Whereas the hAGT C₁₄₅ and U₁₄₅ variants allow DNA repair, the hAGT S₁₄₅ protein is inactive. The chemical structure of MNNG, which causes the conversion of guanosine to O⁶-methylguanosine in DNA, is shown. (B) E. coli­ ΔselA, ΔselB, Δada, Δogt, T7RNAp cells were co-transformed with the UTu components and the hAGT C₁₄₅, UAG₁₄₅ and S₁₄₅ variants. After three selection rounds with 10 μg/ml MNNG pulses and 2 h of recovery in-between, the indicated 10⁻³, 10⁻⁴ and 10⁻⁵ dilutions of cell suspensions were plated on LB agar to check for growth after 24 h.