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. 2011 May 25;6(5):e20152. doi: 10.1371/journal.pone.0020152

Figure 4. Rtt107 phosphorylation is required for its recruitment to the MAT DSB.

Figure 4

(A) The phospho-mutant allele of Rtt107, Rtt107-AQ is not recruited to HO-induced DSBs. A DSB was induced at the MAT locus in a strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7290) or the phospho-mutant version Rtt107-AQ-6HA (CCG7291) is shown. The binding of Rtt107 and Rtt107-AQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviation are shown. A locus on chromosome VI was used as a control. Western blot analysis of Rtt107-6HA and Rtt107-AQ-6HA upon DSB induction is also shown. No band-shift was observed for Rtt107-AQ, suggesting that this protein is not phosphorylated upon DSB induction. (B) The phospho-mimmicking allele of Rtt107, Rtt107-DQ is recruited to HO-induced DSB in the absence of the Mec1 kinase. A DSB was induced at the MAT locus in a mec1Δ strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7558) or the phospho-mimmicking version Rtt107-DQ-6HA (CCG7556). The binding of Rtt107 and Rtt107-DQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.