Effect of I-SceI Endonuclease on Coincidence of Secondary Mutations with Lac⁺ Point Mutations.

aIn this and all of the tables and figures in this paper, stress-induced Lac+ colonies were divided into point mutants (compensatory frameshift revertants) and lac-amplified clones per [14], and only the point mutants were screened for secondary mutations. Because stress-induced lac-amplifications are not associated with secondary mutations (or a HMS) [14], this controls for differential effects of any of the treatments studied on point mutagenesis and amplification. bStrain SMR6277. This strain is a negative control that expresses neither I-SceI endonuclease nor carries the I-SceI cutsite, and so does not make I-SceI-mediated DNA double-strand breaks (DSBs). It is a negative control for the “Enzyme and Cutsite” strain SMR6280 which expresses I-SceI from a chromosomal regulatable promoter PBAD replacing the phage lambda attachment site (Δattλ::PBADI-SceI) and carries an I-SceI site, and makes DSBs. This strain has the PBAD promoter insertion without the I-SceI gene, Δattλ::PBAD, and so is designated “PBAD only”). cStrain SMR6276. This strain is a second negative control for the I-SceI-mediated DSB-producing strain SMR6280. This “Enzyme-only” strain carries the Δattλ::PBADI-SceI expression cassette but no I-SceI cutsite. dStrain SMR6280, with both the chromosomal Δattλ::PBADI-SceI expression cassette and the F'-located I-SceI cutsite, makes I-SceI-induced DSBs near lac[19]. This strain shows greatly increased Lac+ stress-induced mutagenesis ([19] and shown here, Figure 3). e“Mucoid” colonies had a mucoid appearance on minimal M9 glucose plates and did not form colonies on either maltose or xylose MacConkey medium.