Figure 7

Functional analysis of the DNA integrity checkpoint in the slt2 mutant strain. A) Exponentially growing cultures of the wild type (W303-1a) and slt2 (JCY1062) strains were split and incubated for the indicated time in the absence or presence of 200 mM hydroxyurea. Cell cycle distribution of cells was analyzed determining cellular morphology and number of nuclei by microscopy. Cells were classified as unbudded, budded with a single nucleus, budded with two nucleus and dumbble-like cells (cells with a large bud similar in size to mother cells but with a single nucleus), which are indicative of G2/M cell cycle arrest. The presence of dumbble cells with an abnormal elongated bud is also indicated. Graphs represent cell distribution derived from at least three independent cultures. No change in cell distribution was observed in untreated controls at 6 hours incubation time. Pictures correspond to the HU-treated sample at 8 hours incubation time. B) Analysis of the DNA content by flow cytometry of samples from exponentially growing cultures of the wild type (W303-1a) and slt2 (JCY1062) strains incubated for 60 min in the absence or presence of 0.04% MMS. C) Exponentially growing cultures of the wild type (W303-1a), slt2 (JCY1062), mlp1 (JCY1334), slt2 mlp1 (JCY1336), hog1 (JCY1489) and slt2 hog1 (JCY1606) strains were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea or 0,04% MMS. Activation of the chekpoint kinase Rad53 was determined by western analysis. The ponceau staining of the membrane is shown as loading control.