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. 2010 Dec 13;286(6):4931–4940. doi: 10.1074/jbc.M110.195024

FIGURE 1.

FIGURE 1.

Effect of genetic inactivation of NEJ1 on NHEJ and the recruitment of Nej1 to an in vivo DSB. A, cultures of haploid yeast strains, wild-type SLY1A (SLY), SLY1A ΔNEJ1 (SLY Δnej1), and a derivative of SLY1A ΔNEJ1 harboring a plasmid expressing V5-tagged Nej1 (SLY Δnej1 pNej1) were diluted and spotted onto Yeast Extract Peptone-galactose plates to induce DSBs as a consequence of HO endonuclease expression. B, role of NHEJ in the ligation of linearized plasmid DNA after transformation into yeast cells. EcoRV-cleaved and circular plasmid DNAs were transformed into SLY1A (WT), SLY1A ΔDNL4 (Δdnl4), SLY1A ΔLIF1 (Δlif1), and SLY1A ΔNEJ1 (Δnej1) (upper panel) and derivatives of SLY1A containing the empty expression vector (WT pVector) and derivatives of SLY1A ΔNEJ1-harboring plasmids expressing the indicated tagged versions of tagged Nej1 (Δnej1 pFlag-Nej1, pCBP-Nej1, and pNej1-V5) (lower panel). Results are presented as relative transformation efficiencies (ratios of cut versus uncut plasmid) from three independent experiments. Error bars indicate mean ± S.D. C, kinetics of recruitment of V5-tagged Nej1 to a site-specific DSB in SLY1A (WT), SLY1A ΔDNL4 (dnl4-del), SLY1A ΔLIF1 (lif1-del), and SLY1A ΔyKU70 (ku-del) strains were measured by chromatin immunoprecipitation as described under “Experimental Procedures.” Data represent the mean ± S.D. of three or more independent experiments.