Figure 5.

MRN is required for NHEJ repair of free DNA fragments in vivo. (A) MRN and ctp1 null mutants are proficient in single molecule plasmid NHEJ repair. NHEJ plasmid repair assays were performed in strains from the indicated genotypes. Results are plotted as the ratio of the number of transformants obtained when transforming with linearized plasmid over those transformed with uncut plasmid. (B) MRN is required tether independent DNA fragments for NHEJ repair in vivo. Strains from the indicated genotypes were co-transformed with KpnI digested plasmid carrying resistance to G418 and an equimolar amount of a DNA fragment encoding for LEU2. After co-transformation, results were scored as the number of colonies in double selection media. The values plotted represent the ratio of colonies grown in double selection media over the ones obtained in parallel transformations using the uncut pKan1 plasmid. (C) The nuclease and tethering activities of Rad32 are dispensable for in vivo single plasmid NHEJ repair. Single plasmid repair assays were performed in strains from the indicated genotypes as in Figure 5A. (D) The dimerization domain is required for tethering unlinked DNA fragments for NHEJ repair. Nuclease domain of Rad32 is differentially required, whereas rad32-D65N nuclease dead is proficient in NHEJ, both rad32-H68S and rad32-H134S mutants are unable to join free DNA ends. Split molecule plasmid repair assays were performed in strains from the indicated genotypes as in Figure 5B. Results for both the single and split molecule plasmid assays are presented as the average of at least three experiments for each strain. Error bars represent 2 × s.e.m.