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

Figure 2. Rtt107 phosphorylation upon DNA damage requires Mec1 kinase and the Smc5–Smc6 complex.

Figure 2

(A) Phosphorylation of Rtt107 upon DNA damage is dependent on the ATR kinase, Mec1. Logarithmically growing cultures of Wt (CCG6886), mec1Δ (CCG6887), rad53Δ (CCG6888) rad9Δ (CCG6683), chk1Δ (CGG6890) and mrc1Δ (CCG6720) carrying myc tagged Rtt107 were treated with 0.3% MMS, 50 µM HU or 50 µg CPT for 90 minutes. An untreated control was also used. Samples were then fixed with RIPA buffer. Indicated extracts were then run on SDS-PAGE gels (as in Fig. S1). Immunoblots were probed with anti-Myc antibodies. Logarithmically growing cultures of Wt (CCG6983) containing a Myc-tagged version of Rtt107, and a galactose-inducible HO endonuclease were grown at 30°C in YP raffinose. Cells were then split and half transferred to galactose (cut), while the other half were grown in the absence of galactose (uncut). Samples were then fixed with RIPA buffer at the indicated timepoints. Extracts were then fractionated on SDS-PAGE gels (as in Fig. S1). Immunoblots were probed with anti-Myc antibodies. (B) Logarithmically growing cultures of smc6–9 (CCG6365) carrying myc tagged Rtt107 were treated with 0.3% MMS for 90 minutes. The experiment was conducted at 25°C and 37° in order to detect phosphorylation at permissive and non-permissive temperatures. To inactivate the smc6–9 allele, cultures were grown overnight at 25°C and then transferred to 37°C for 2 hours, prior to the addition of MMS. Samples were processed as in A.