Figure 2.

VDE-initiated crossovers at most loci are MutLγ-independent. (A) Strategy for detection of VDE-initiated COs and NCOs. A cartoon of the VRS and VRS-103 inserts is shown, illustrating: white box—VRS sequences; blue arrows—HindIII restriction sites; green lines—sequences shared between the two inserts, with ARG4 coding sequences shown as a green arrow; green box—sequences used for Southern blot probes. Digestion with HindIII and PI-SceI (VDE) distinguishes parental (P1 and P2), CO and NCO products. VDE-cut inserts are not distinguished from parent P1 in these digests, but can be distinguished in digests with HindIII alone (Medhi et al. 2016). (B) Representative Southern blot containing DNA from strains with inserts at RIM15. (C) VDE-initiated COs in MLH3 and mlh3∆ cells. CO frequencies, average signal of CO1 and CO2 for 8 and 9 h samples from three independent experiments for inserts at HIS4 and from two independent experiments for inserts at all other loci. Data for inserts at URA3 and for two experiments with inserts at HIS4 are from Medhi et al. (2016). (D) fraction of COs that are MutLγ-independent (ratio of CO frequencies in mlh3∆ vs. MLH3), plotted as a function of CO frequencies in MLH3 strains. CO frequencies in MLH3 and mlh3∆ differ significantly only for inserts at HSP30 and HIS4 (adjusted p values of 0.003 and 0.0001, respectively) (E,F) VDE-initiated NCOs, details as in (B) and (C); frequencies in MLH3 and mlh3∆ do not differ significantly at any locus (adjusted p values ≥ 0.05). Error bars in all panels denote standard deviation. See Figure S2C for summary plots with CO and NCO values for all genotypes.