Fig. 4

Hyper-activation of the 9-1-1 axis enhances Rad53 activation even at low levels of Mec1 recruitment. a A covalent Ddc1-Rad9 interaction results in enhanced Rad9 recruitment and blocks Mec1–Ddc2 kinase recruitment to a double-strand break (DSB). Wild-type (WT) cells and DDC1-RAD9-fusion cells (same construct as used in Fig. 1f–g) were arrested in M phase and analysed at indicated times. Depicted are chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) measurements of Mec1–Ddc2 recruitment (upper panels, ChIP directed against Ddc2–3FLAG using an anti-FLAG antibody) and Rad9 recruitment to a DSB (lower panels, ChIP directed against Rad9 or the fusion which both carry a 3FLAG tag for detection). b Rad53 activation in response to a DSB is strongly enhanced in cells expressing a DDC1-RAD9-fusion construct. Western blot analysis of Rad53 activation at indicated times after DSB induction, strains as in a. Cdc48 served as loading control (lower panel). c A covalent Rad9-Dpb11 fusion protein enhances Rad9 recruitment to the DSB and blocks DNA end resection. WT cells and cells expressing RAD9-dpb11∆N (lacking BRCT 1+2 of Dpb11 which normally bind to Rad9) were arrested in M phase and analysed at indicated time points. ChIP-qPCR measurements of DNA resection (replication protein A (RPA), upper panels) and Rad9 recruitment to a DSB (lower panels). To measure Rad9 recruitment, Rad9-dpb11∆N-fusion and Rad9, respectively, were tagged C-terminally with a 3FLAG tag. d Rad53 activation in response to a DSB is enhanced in RAD9-dpb11∆N-fusion cells. Western blot analysis of Rad53 activation at indicated times after DSB induction, strains as in c, Cdc48 serves as loading control