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Fig. 5. Myc-Mec1 focus formation and recruitment in rad24, rfa1-t11 and rad51 mutants. (A) Myc-Mec1 localisation was determined by IF in JKM179 derivatives expressing Myc-tagged Mec1 (GA1529, shown in the insets in the rad9 panels), or in strains deleted for rad9, for rad24 or both, after 0 and 4 hours of HO induction on galactose. IF was conducted as in Fig. 1B. Values correspond to the percentage of nuclei exhibiting a single Mec1 focus. (B) Myc-Mec1 ChIP at the HO cleavage site as described in Fig. 1C using the rad9 rad24 double-deletion strain or the wild-type (WT) JKM179 derivative. (C) The percentage of ssDNA at the HO1 site was measured, after HO induction, by QAOS on total DNA from the JKM179-derivative GA1529 (WT), or from JKM179 with null alleles of rad51, rad9 rad24 or rfa1-t11. (D) IF as in Fig. 1B on the wild-type JKM179 derivative (GA1529) and isogenic strains bearing rfa1-t11 (GA2158) or a rad51 deletion (GA2163). Values correspond to the percentage of nuclei exhibiting a single bright Mec1 focus above the background of diffuse Mec1. Note that absence of a focus does not necessarily mean that no Mec1-Ddc2 binds the DSB but rather that the level is below the threshold of detection by IF. (E) Myc-Mec1 ChIP as described in Fig. 1C on JKM179 carrying either the rfa1-t11 mutation or a rad51 deletion. Results are shown for the HO2 probe only, although all probes showed analogous results. Bar, 1 µm.
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