Analyses of damage-inducible Sfr1 and Swi5 foci. (A) The EGFP-Sfr1 signals and chromosomal DNA (stained with Hoechst 33342) of exponentially growing wild-type cells (YA848) were observed with a fluorescent microscope. (B) Rhp51 (left, YA990), Swi5 (middle, YA979) and Sfr1 (right, YA848) assemble following UV irradiation. Exponentially growing cells were irradiated with 100 J/m² UV light. After incubation for 3 h at 30°C, cells were observed by using a DeltaVision microscope system. Note that focal signals of Swi5-EGFP were analyzed in an swi2Δ background to abolish the accumulation of spontaneous foci at heterochromatin. The graph shows percentages of cells with foci before (−) and after (+) UV irradiation. (C) Sfr1 assembly is dependent on Rhp51. EGFP-Sfr1 focal signals are not detected in cells (YA986) with the rhp51Δ mutation 3 and 6 h after UV irradiation.

(A) Rhp51-ECFP assembles at sites of HO-induced single DSBs (YA1083). (B) Swi5 and Sfr1 colocalize with Rhp51 following UV irradiation. Top, cells with Swi5-EGFP and Rhp51-ECFP fusion proteins (YA1263); bottom, cells with EGFP-Sfr1 and Rhp51-ECFP fusion proteins (YA1213). Each inset is a magnified image of the same nucleus. In the merged images, green indicates Swi5-EGFP or EGFP-Sfr1 and magenta indicates Rhp51-ECFP signals. Each inset is a magnified image of the same nucleus. Note that focal signals of Swi5-EGFP were analyzed in an swi2Δ background to abolish the accumulation of spontaneous foci at heterochromatin. (C) Spontaneous Swi5 foci are independent of rhp51⁺ and the DSB at the smt locus within the mat1 region. The strains used were rhp51⁺ smt-0 (YA961) and rhp51Δ smt-0 (YA984).

Localization of Swi2 and Swi5 proteins. (A) Swi5 localizes to the nucleus in two patterns. The diffuse nuclear localization of Swi5 is dependent on sfr1⁺, whereas focal formation requires swi2⁺. The Swi5-EGFP signals of exponentially growing cells were observed by fluorescence microscopy. The strains used were wild-type h⁹⁰ (YA598), sfr1Δ (YA763), swi2Δ (YA673), swi2Δ sfr1Δ (YA765) and swi6Δ (YA752). (i)–(v) Images were obtained by using a conventional fluorescence microscope (Nikon ECLIPSE E800). (vi) Image was obtained by using a DeltaVision microscope system as described in Materials and methods. The bar indicates 10 μm. (B) Neither the swi2Δ nor sfr1Δ mutation affects the level of Swi5-EGFP expression. Total protein extracts (10 μg) from the indicated strains were separated by SDS–polyacrylamide gel electrophoresis and subjected to immunoblot analysis with an anti-GFP antibody (JL-8, Clontech). The same strains were used as in (A) and the untagged strain was YA254. (C) Swi2 forms a few spontaneous foci in the nucleus. Swi2-EGFP signals of exponentially growing cells were observed by a DeltaVision microscope system. The strains used were wild type (YA820), swi5Δ (YA1247) and swi6Δ (YA1261). (D) Swi5 foci colocalize with Swi6 foci. A strain (YA756) with EGFP-tagged swi5⁺ and ECFP-tagged swi6⁺ was examined as in (C). In the merged image, green indicates Swi5-EGFP and magenta indicates Swi6-ECFP signals. Each inset is a magnified image of the same nucleus. The graph shows percentages of cells with unlocalized (gray bars) and colocalized (open bars) foci. X-axis gives the number of Swi5 foci in each cell. (E) Swi2 foci colocalize with Swi6 foci. A strain (YA1292) with EGFP-tagged swi2⁺ and ECFP-tagged swi6⁺ was examined as in (C). In the merged image, green indicates Swi2-EGFP and magenta indicates Swi6-ECFP signals. Each inset is a magnified image of the same nucleus. The graph shows percentages of cells with unlocalized (gray bars) and colocalized (open bars) foci. X-axis gives the number of Swi2 foci in each cell.

(A) In rhp54Δ cells (YA1240), EGFP-Sfr1 foci form spontaneously under normal growth conditions, but much more foci of stronger intensity are observed following UV irradiation (120 J/m²). Foci persisted for at least 26 h after UV irradiation. (B) Time course of foci formations of Swi5 and Sfr1 after UV irradiation (120 J/m²). Strains used were YA848 (EGFP-Sfr1), YA979 (Swi5-EGFP in swi2Δ), YA1325 (Swi5-EGFP in swi2Δ rhp54Δ) and YA1240 (EGFP-Sfr1 in rhp54Δ).

Rhp57- and Swi5-dependent subnuclear accumulation of Rhp51 upon UV irradiation. (A) A multicopy plasmid expressing rhp51⁺ suppresses the UV sensitivity of the swi5Δ rhp57Δ double mutant. Wild-type (YA119), rhp51Δ (T3) and swi5Δ rhp57Δ (YA250) cells carrying the vector alone (pSP102) or a vector expressing rhp51⁺ (pSP192) were examined for UV sensitivity. The data represent the average of the results from three independent experiments and standard deviations are shown by error bars. (B) Rhp51 accumulation in wild-type and mutant cells was detected by immunostaining with the anti-Rhp51 antibody. Wild-type (YA119), swi5Δ (YA177), rhp57Δ (T5), swi5Δ rhp57Δ (YA250) and rhp51Δ (T3) cells were examined without UV irradiation or 3 h after UV irradiation (200 J/m²). (C) Mean percentage of mutant cells showing Rhp51 foci at 3 h post-irradiation. Frequencies were determined as described in Materials and methods.