Fig. 1
From: Quantitative sensing and signalling of single-stranded DNA during the DNA damage response
Differential regulation of DNA damage checkpoint effectors γH2A and Rad53. a Different amounts of Mec1–Ddc2 kinase phosphorylate H2A with similar efficiency. Wild-type (WT) and long-range resection-deficient exo1∆ sgs1∆ strains were arrested in M phase by nocodazole treatment, a non-repairable double-strand break (DSB) at MAT was induced by Gal-HO expression and protein recruitment was measured by chromatin immunoprecipitation (ChIP) at indicated times. Upper panel: fold enrichment of a given locus in a replication protein A (RPA) ChIP relative to undamaged control loci. Second panel: Mec1–Ddc2 kinase recruitment detect by ChIP against Ddc2–3FLAG. Third panel: H2A-S129 phosphorylation (γH2A phosphorylation). Lower panel: Rad53 kinase recruitment. b The checkpoint kinase Rad53 is activated in a resection-dependent manner. Western blot detecting the phosphorylation-dependent shift of activated Rad53 with an anti-Rad53 antibody and an anti-Cdc48 loading control. The samples were obtained at indicated time points after Gal-HO induction in M phase-arrested cells. c γH2A phosphorylation around a DSB is long-range resection independent. Overlay of γH2A ChIP-seq profiles around a DSB at MAT 4 h after DSB induction in WT cells (blue) and exo1∆ sgs1∆ cells (purple). Enrichment plotted relative to the whole genome average. d, e γH2A phosphorylation induced by a single DSB is not influenced by the cell cycle, while Rad53 phosphorylation is. ChIPs from WT cells as in a, but cells were arrested either in G1 by alpha-factor treatment (left panels) or in M phase by nocodazole treatment (right panels). e Western blot analysis of Rad53 activation as in b, but with G1- and M phase-arrested cells. f The DNA end resection regulators Fun30 and Rad9 do not affect γH2A phosphorylation. A DSB in M phase was induced in WT cells, hyper-resecting strains (rad9∆, DDC1-FUN30 fusion) or resection-inhibited strains (fun30∆, DDC1-RAD9 fusion). Resection (left panel, ChIP against RPA) and γH2A phosphorylation (right panel) were measured at indicated time points. g Overlay of ChIP-quantitative PCR (qPCR) traces of RPA and γH2A after 4 h of DSB induction from f in WT cells (blue), hyper-resecting cells (green) and resection-inhibited cells (red)
