Extended Data Fig. 2: Cell cycle analysis of a MEC1 mutant with constitutive activity.

a, Western blot analysis of phospho-H2A (pS129) (top) and Rad53 (bottom) in wild-type strain PY405, with either MEC1 or mec1-F2244L. Cells were arrested in G1 phase with alpha-factor, or arrested in G1 phase with alpha-factor, and released into S phase with 200 mM of hydroxyurea for the indicated time. Ponceau staining of the blot is shown below. b, mec1-F2244L suppresses the growth defect of activator-defective yeast. In the experimental scheme, the extreme defects of the activator-defective strain were initially suppressed by a plasmid-borne copy of wild-type DNA2, containing the URA3 gene as selectable and counterselectable marker. Thus, strain MEC1 tel1Δ ddc1Δ dna2Δ (PY270) contains three plasmids: p(DNA2 URA3), p(dna2-WYAA TRP1), and either vector or p(mec1-x LEU2). The strains were grown on media lacking Trp and Leu (left), or on 5FOA-containing media (right) that only permits growth if the p(DNA2 URA3) plasmid is lost. The data indicate that mec1-F2244L allows cell growth without p(DNA2 URA3), therefore suppressing the growth defect of the activator-defective strain. c, Ponceau staining of the extracts used for the blots in Fig. 2g. d, Constitutively active mec1-F2244L progresses slowly through S phase. Strain PY406 containing p(MEC1 LEU2) (blue) or p(mec1-F2244L LEU2) (red). Cell cycle distribution was measured for (a) asynchronous cells; (b) alpha-factor arrested G1 cells; (c) G1 arrested cells treated with 4NQO for 30 min; (d, e, f) G1 arrested cells released into fresh YPD for 5, 30, and 60 minutes; (g) G1 arrested cells released into fresh YPD containing 200 mM hydroxyurea for 60 minutes, (h, i, j) example of gating strategy shown for plot (a) p(MEC1 LEU2) asynchronous cells.