DNA damage sensitivity of rad50-d cells is suppressed by deletion of pku70⁺. The sensitivities to γ rays, HU, MMS, and UV light of rad50-d cells and rad50 pku70 double mutants. The percent survival of wild-type cells, JY741 (•), rad50-d, KT002 (▴), and rad50 pku70 double mutants, KT152 (▵) is plotted against the dose of γ rays (A) or UV light (B) or is plotted against time in 0.02% MMS (C) or 20 mM HU (D). Standard deviations are shown by error bars.

Suppression of DNA damage sensitivity occurs upstream of Rad51. Cell survival frequencies on 0.002% MMS-containing plates versus control plates for wild-type cells (JY741), rad50-d (KT002), rhp51-d (KT00c), rhp51-d pku70-d (KT10c5), pku80-d (KT090), rad50-d pku80-d (KT192), lig4-d (KT1a0), and rad50-d lig4-d cells (KT1a2). Standard deviations are shown by error bars (for two to four independent experiments).

rad50 exo1 double mutants become more sensitive to DNA damage than each single mutant. (A) The sensitivities to γ rays of wild-type cells, rad50-d, exo1-d, rad50-d exo1-d, and rhp51-d cells. The percent survival of wild-type cells (700), rad50-d (701), exo1-d (702), rad50-d exo1-d (703), and rhp51-d cells (704) is plotted against the γ ray dose. (B) Cell survival frequencies on 0.002% MMS-containing plates versus control plates for wild-type cells (JY741), exo1-d (KΤ00g), rad50-d (KT002), rad50-d exo1-d (KΤ02g), and pku70-d rad50-d exo1-d (KΤ12g5) cells. Standard deviations are shown by error bars (for two to four independent experiments). (C) The MMS sensitivity of wild-type cells (119), rad32-d (324), rad32-d pku70-d (315), rad32-d exo1-d (403), pku70-d rad32-d exo1-d (407), pku70-d (319), exo1-d (302), and exo1-d pku70-d (394) cells was assayed by the spot test. The cells were grown in YEA (0.5 × 10⁷ cells/ml), serially diluted (1:10) with sterilized water, and spotted (4 μl of each dilution was spotted onto the MMS plates).

Exo1 and rad50 function independently upstream of Rad51. (A) Indirect immunofluorescence microscopy of wild-type cells (700), rad50-d (701), exo1-d (702), and rad50-d exo1-d cells (703) at 1 h after irradiation with 500 Gy of ionizing radiation (8). A cross-reacting human anti-Rad51 antibody was used to detect Rhp51. (B) Mean percentage of various mutant cells showing Rhp51 foci at 1 h postirradiation.

The roles of Rad50 and Ku70 at telomere ends in taz1-d cells. The single-stranded overhangs were detected by in-gel hybridization. (A) Lane 1, taz1-d (KT110); lane 2, rad50-d taz1-d (KT021); lane 3, rad32-d taz1-d (KT116); lane 4, pku70-d rad50-d taz1-d (KT0215); lane 5, pku70-d rad32-d taz1-d (KT1165); lane 6, pku70-d rad50-d exo1-d taz1-d (KT121g5); lane 7, pku70-d rad32-d exo1-d taz1-d (KT016g5); lane 8, dsDNA control; lane 9, ssDNA control. (B) Lane 1, taz1-d trt1-d (KT117); lane 2, wild-type cells (JY741); lane 3, dsDNA control lane 4, ssDNA control. (C) Lane 1, taz1-d (KT110); lane 2, rad32-d taz1-d. (KT116); lane 3, rad32-D25A taz1-d (KT0106M1); lane 4, rad32-D25A taz1-d (KT0106M2); lane 5, dsDNA control; lane 6, ssDNA control. Genomic DNAs were digested with EcoRI and separated by electrophoresis on a 0.5% agarose gel. Then the gel was dried and hybridized with ³²P-labeled C-rich (top panel) or G-rich (middle panel) probe. To detect double-stranded telomere DNAs, the gel was treated with denaturant and reprobed with C-rich probe (bottom panel). As a control, a linearized telomeric DNA-containing plasmid was used for double-stranded telomere detection. For the single-strand control, the same linearized plasmid, which was denatured, was used (see Materials and Methods). Telomere DNA is indicated by arrows. (D) Rad32-D25A binds to Rad50 in immunoprecipitation assay. TAP-tagged Rad50 was precipitated with IgG-conjugated magnetic beads from a lysate of TAP-rad50 rad32-Myc (KTt2T6M) cells (lanes 3 and 8) and a lysate of TAP-rad50 rad32-D25A-Myc (KTt2T6MM1) cells (lanes 4 and 9), respectively. The immunoprecipitates (IP with IgG) were examined by Western blot (Western) analysis with anti-Myc antibody (anti-Myc). The protein A tag is detected with anti-protein A antibody (anti-Protein A). TAP-rad50 cells (435); lanes 5 and 10, rad32-Myc (KTt6M) cells; lanes 1 and 6, rad32-D25A-Myc (KTt6MM1) cells; lanes 2 and 7 were used as controls. The same levels of proteins were detected in the whole-cell extract (WCE). (E) Rad32-D25A is bound to telomere DNA in ChIP assay. Untagged wild-type control cells (JY741), rad32-Myc (KTt6M) cells, and rad32-D25A-Myc (KTt6MM1) cells were used. PCRs were performed on whole-cell extract (WCE) and on chromatin immunoprecipitates (IP with anti-Myc) by using primers to amplify a telomere DNA (telomere) and primers to amplify DNA from the ade6⁺ gene (ade6). M represents a DNA maker.

Models. (A) Hypothetical model for DSB processing by Rad50 complex and Exo1. DSB ends are processed mainly by the Rad50-Rad32 complex. In the absence of Rad50-Rad32 complex, DNA ends can be processed by Exo1, but Ku heterodimer has to be removed from the break site to allow end processing. The Cdc13 function may act through the Exo1 pathway, in parallel to Rad50 (8). (B) Hypothetical model for degradation of C-rich strand at telomere ends in taz1-d cells. Telomere ends in taz1-d cells are processed mainly by the Rad50-Rad32 complex. Because the nuclease domain is not required for this process, we assume that the Rad50 complex recruits an unknown nuclease. In the absence of Rad50 complex, DNA ends can be processed by unknown nucleases, but Ku heterodimer has to be removed from the break site to allow end processing.