Fig. 9.
UmuC loop 2 variants do not confer sensitivity to UV radiation and are proficient for mutagenesis. (A) UV (25 J/m2)-induced mutation frequency of selected variants in plasmid pGY9738 (umuD′C) in strain GW8017 (ΔumuDC). The wild-type plasmid was pGY9738, and the empty vector was pGB2. Frequencies were as follows: for the empty vector, induced mutants, 7.66 × 10−7; spontaneous mutants, 3.36 × 10−7; mutation frequency, 4.31 × 10−7; for the wild type, induced mutants, 1.85 × 10−5; spontaneous mutants, 6.48 × 10−6; mutation frequency, 1.21 × 10−5; for K50A, induced mutants, 3.28 × 10−5; spontaneous mutants, 2.96 × 10−6; mutation frequency, 2.98 × 10−5; for M51A, induced mutants, 1.71 × 10−5; spontaneous mutants, 3.00 × 10−6; mutation frequency, 1.41 × 10−5; for G52A, induced mutants, 3.38 × 10−5; spontaneous mutants, 6.36 × 10−6; mutation frequency, 2.74 × 10−5; for D53A, induced mutants, 1.16 × 10−5; spontaneous mutants, 3.32 × 10−6; mutation frequency, 8.29 × 10−6; and for P54A, induced mutants, 3.77 × 10−5; spontaneous mutants, 7.22 × 10−6; mutation frequency, 3.05 × 10−5. (B) Loop 2 variants do not confer sensitivity to UV radiation. Assays were performed with pGY9738 plasmid and the following derivatives in PB102: pGY9738-K50A (umuD′C K50; ×), pGY9738 (umuD′C wild type; ■), pGY9738-G52A (umuD′C G52A; ○), pGY9738-M51A (umuD′C M51A; □), pGY9738-P54A (umuD′C P54A; ♢), pGY9738-D53A (umuD′C D53A; ●), and pGB2 (empty vector; ◆). (C) Immunoblot showing steady-state levels of UmuC expressed from variant umuDC plasmids (GW8017). The wild-type plasmid was pGY9738, and the empty vector was pGB2.