Fig. 2: Protein engineering increases activity of RAG-mediated, homology-assisted recombination of split antibiotic resistance gene.

a Diagram of pY112-CJA-UP-H20 recombination target plasmid. Triangles represent RSSs. Cutting by RAG1/2 leads to homology-assisted DNA repair of the G418R gene that confers resistance to the antibiotic G418. b Yeast strains were engineered with both RAG1 and RAG2 genes (either full-length, core, or core NLS variants) and optionally HMGB1. After 4-d induction in SG-Leu media, cell cultures were plated on Leu, G418 plates and colonies were counted after outgrowth. c G418R recombination with yeast strains assessing the combination of full-length RAG2 with RAG1core. Cells were plated after a 4-d induction. d G418R recombination with alternate truncations of RAG1 that include more regions of the full-length protein than the initial RAG1core construct (which contains amino acids 383-1006). All strains include full-length RAG2 and HMGB1. Cells were plated after a 4-d induction. C = core protein, F = full-length protein, N-C = core protein with N-terminal nuclear localization signal, NLS = nuclear localization signal, CFU = colony forming unit. In (b–d) strains that appear in multiple plots are highlighted with a color, and data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated with a one-way ANOVA and Tukey test (ns = not significant, **p < 0.01, and ****p < 0.0001). From left to right, the highlighted p values are, in (b), p = <0.0001 and 0.0042; in (c), p = <0.0001 and <0.0001; and in (d), p = <0.0001 and 0.0148. Source data are provided in the Source Data file.