Apai sacii

Mutations in the specific motifs of RadA were constructed by site-directed mutagenesis (Quikchange, Stratagene) of radA on a high-copy pBS SK-derived plasmid, pSTL307 (Beam et al., 2002 (link)). Primers used to generate the site-directed mutants included: K108R: 5′ AGG GTT ACC GCC AAT CAG AAT GG 3′; K258A: 5′ CAA GGT GCG AAA ACG GGA GTC G 3′; S372A: 5′ TTA CCT TCA CGC CGC CGA CCA CGT TCA 3′. RadA-C28Y was generated by Polymerase Chain Reaction (PCR) amplification of the radA gene derived from the radA100 strain SR776 (Diver et al., 1982 (link)) as described previously (Beam et al., 2002 (link)). The sequence of the radA alleles were confirmed by DNA sequence analysis of the entire gene and alleles were transferred to the low-copy plasmid pWSK29 (Wang and Kushner, 1991 (link)) by ApaI SacII digestion and ligation with T4 ligase (New England Biolabs). Mutant alleles with truncation of the C-terminal Lon domain were constructed by PCR amplification with Pfu polymerase (Stratagene) and the appropriate primers, followed by HindIII digestion and ligation. Primers included 5′ GGG GGG GAA GCT TTT ATT ACT GTT CGG TCA TCG CGA AGA C 3′ and 5′ GGG GGG GAA GCT TTG AGA TAT ACG GAG GGA GAT ATG TCG TCA T 3′ for RadAΔ275–460 or 5′ GGG GGG AAG CTT TTA AGC CAG CGA ACC ATC TTT GGT TAC G 3′ and 5′ GGG GGG AAG CTT CGA TAC CGT CGA CCT CGA GGG GGG GCC CGG TA 3′ for RadAΔ228–460.