Constructing Lesion-Containing Genomes

The lesion-containing as well as the lesion-free control and competitor genomes were prepared, following the previously published procedures^{20 (link)}. Briefly, the parental vector was digested with Nt.BstNBI to generate a gapped plasmid, followed by removal of the resultant 25-mer single-stranded ODN through annealing with a 25-mer complementary ODN that is in 100× excess. The gapped plasmid was then isolated from the mixture by using 100 kDa cutoff ultracentrifugal filter units (Millipore). The purified gapped plasmid was annealed with a 5′-phosphorylated 13-mer lesion-free ODN (5′-AATTGAGTCGATG-3′) and a 5′-phosphorylated 12-mer lesion-carrying or lesion-free control ODN, 5′-ATGGCGXGCTAT-3′ (X = α-dN, x-dN, or dN), followed by incubation with T4 DNA ligase and ATP in the ligation buffer (Figure 2).