Figure 3.

The involvement of NIPBL in NHEJ is evolutionarily conserved. (A and B) WT cells, cells expressing a temperature-sensitive allele of the yeast NIPBL orthologue SCC2 (scc2-4), and cells with a deletion of lig4Δ, either alone or in combination with the scc2-4, were arrested in G1 by addition of A-factor for 2 h and subsequently incubated at a restrictive temperature (32°C) for 30 min. A site-specific DSB was then induced by the galactose inducible HO enzyme for 60 min, as indicated, during continuous G1 arrest at a restrictive temperature. (A) The cell cycle distribution was determined by FACS analysis before addition of A-factor and on G1-arrested cells, 2 h after addition of A-factor. (B) After DSB induction, cells were plated in the absence of galactose at equal starting concentrations and in 6 10-fold subsequent dilution steps (indicated by triangle). Plates were left to recover for 72 h at 30°C (semipermissive temperature for scc2-4). One representative experiment from ≥3 is shown.