Model for transformation of trp5 G148T strains with TS or NTS oligos. (A) An oligo (orange) anneals at the replication fork, creating a mismatch (asterisk and orange arrow), and is extended to form an Okazaki fragment. MMR will remove the oligo, but if the oligo escapes MMR it will be incorporated into the genome. (B) An oligo (orange) is introduced into a cell and anneals with chromosomal DNA (blue). TAA is the NTS sequence of the mutant glutamic acid codon that must be mutated to GAA to give Trp⁺ revertants in the trp5 G148T strains. At the end of the first round of replication, Trp⁺ mRNA can be transcribed from the TS oligo, but RNA produced from transformation with a NTS oligo will still be Trp⁻. MMR correction operating outside the context of the replication fork after the first round of replication could result in two alternatives for each oligo, including Trp⁺ mRNA from an NTS oligo and Trp⁻ mRNA from a TS oligo.

NTS but not TS oligos require a second round of replication for transformation in MutSα-deficient (msh6) strains. (A) trp5 G148T msh6 (Lys⁻, Trp⁻) was transformed with a TS or NTS oligo and the number of Trp⁺ revertants arising on selective plates determined after different growth times in rich medium. (B) One-hundred microliters of a culture of trp5 G148T growing in YPAD was placed in 5 mL of synthetic dextrose (SD) medium lacking lysine, tryptophan, or medium supplemented with the indicated fraction of the normal concentration of tryptophan (20 μg/mL). At the indicated time points, A₆₀₀ was measured, with each point being the average of three measurements.

MMR circumvents the requirement of a second round of replication for NTS oligo transformation. Trp⁺ revertants resulting from transformation with either NTS or TS oligos were determined after 45-min (short) or 2-h (long) recovery time in rich medium. Trp⁺ revertants obtained at the short recovery time are shown as a percentage of revertants obtained at the long recovery time. Error bars indicate the SD from three or more experiments. NTS oligos create a G-A mismatch; TS oligos create a C-T mismatch.

The 8-oxodGTP transformation displays what appears to be adaptive mutation in wild-type strains and a strict requirement for a second round of replication in msh6 strains. (A) Transformation of trp5 G148T wild-type cells with dGTP or 8-oxodGTP plated after a short recovery time (15 min). Shown is the number of Trp⁺ revertants counted after the indicated days of incubation on selective plates. (B) Transformation of trp5 G148T strains with 50 nmol 8-oxodGTP. One-half of each transformation was plated after a 15-min recovery time and one-half plated after a 2-h recovery time. The number of colonies on each plate was determined multiple times on the indicated days. Shown at each time point is the percent of transformants obtained for the indicated incubation and recovery time compared with the number of transformants obtained in that experiment after 15 d of incubation for the 2-h recovery time. Error bars indicate the SD from four experiments for each strain.