The Dia2 protein binds replication origins. (A) Chromatin immunoprecipitation was performed on the Dia2-18×myc–tagged strain and an untagged strain. After formaldehyde cross-linking, cells were harvested and lysates prepared. Equal amounts of total protein were subjected to immunoprecipitation with anti-myc antibodies. Chromatin bound during the reaction was isolated and cross-links were reversed before PCR analysis. Primers specific to ARS305 (an early-firing origin), ARS603 (a late-firing origin), and ATP11 (a nonorigin control) were used for PCR. (B) Skp1 and Cdc53 bind origins. Chromatin immunoprecipitation was performed as in A using anti-Skp1 and anti-myc antibodies in a WT strain carrying a Cdc53-myc–tagged vector. (C) Dia2 binds replication origins in a cell cycle–dependent manner. Chromatin immunoprecipitation was performed as in A except that cells were arrested in G1-, S-, and M-phase by treatment with alpha factor, HU, and nocodazole, respectively.

dia2 mutants are hypersensitive to DNA damage. (A) The dia2 cdc13-1 strain exhibits greater loss of viability than the cdc13-1 strain. The indicated strains were incubated at 37°C for the indicated times. Cells were then plated on rich media and incubated at room temperature until colonies formed. Colony counts are expressed as a percentage using the 0 time point as 100%. Error bars indicate the SD of at least three experiments. (B) dia2 mutants are sensitive to MMS. Serial dilutions of the indicated strains were spotted onto selective media containing the indicated amounts of MMS and incubated at either 25 or 30°C.

Deletion of CLB5 and CLB6 alleviates the premature S-phase in dia2 cells and leads to decreased S-phase damage. (A) The early S-phase entry in dia2 is dependent on Clb5 and Clb6. dia2, clb5 clb6, and dia2 clb5 clb6 cells were arrested in G1 with alpha factor and then released from arrest at 25°C. Samples were taken at the indicated time points and used for flow cytometry. (B) The S-phase damage in dia2 cells is dependent on Clb5 and Clb6. The number of Rad52 foci and bud morphology as a measure of cell cycle stage were counted in cells treated as in Figure 6. At least 150 cells were counted per strain. Error bars indicate the SD of at least three experiments.

The dia2 mutant accumulates Rad52-YFP foci. (A) WT, dia2, and mec1 sml1 strains containing Rad52-YFP were grown to log phase in rich media and then examined by fluorescence and DIC (differential interference contrast) microscopy. Arrows point to examples of foci. Equal exposure times were used for all strains. Scale bar, 5 μm. (B) The dia2 mutant accumulates Rad52-YFP foci in S- and M-phases. The number of Rad52 foci and bud morphology as a measure of cell cycle stage were counted in cells treated as in A. At least 150 cells were counted per strain. Error bars indicate the SD of at least three experiments. (C) The ΔF-box mutant accumulates Rad52 foci in S- and G2/M-phases. The number of Rad52 foci and bud morphology as a measure of cell cycle stage were counted in cells treated as in A, except that the cells were grown in selective media to maintain the appropriate plasmid. At least 150 cells were counted per strain. Error bars indicate the SD of at least three experiments.

Dia2 is an F-box protein required for normal growth. (A) Diagram of Dia2 domain structure. Structure adapted from (Patton et al., 1998; Katayama et al., 2002). Modified forms described in the text are shown in a and b. (B) Recombinant Dia2 assembles with SCF components. Insect cells were coinfected with HA₃-tagged Dia2 baculovirus and Flag-tagged Skp1 or Myc-tagged Cdc53 baculoviruses. After 40 h, cells were harvested and lysates were subjected to immunoprecipitation with anti-Flag or anti-myc antibodies. Immunoblots were probed with anti-HA antibodies. I, input; IP, immunoprecipitate. (C) Dia2 assembles with SCF components in yeast cells. Skp1 was immunoprecipitated using anti-Skp1 antibodies (Skowyra et al., 1997) from the indicated strains. Immunoblots were probed with anti-myc and anti-Skp1 antibodies. Because of significant differences in signal intensity between Dia2 and Cdc53, two separate blots are shown. (D) The dia2 deletion exhibits a slow growth phenotype at room temperature. Colonies from tetrad dissection of DIA2/dia2 diploid induced to undergo meiosis are shown. Arrows point to representative small dia2 colonies, whereas large colonies are DIA2.

The dia2 mutant exhibits S-phase defects. (A) SCF mutants are HU-sensitive. Serial dilutions of WT, dia2, skp1-11, and cdc53-1 strains were spotted onto rich media containing indicated amounts of HU and grown at 30°C. (B) The dia2 mutant enters S-phase earlier than WT. WT and dia2 cells were arrested in G1 with alpha factor and then released from arrest at 25°C. Samples were taken at the indicated time points and used for flow cytometry. The percentage of S-phase cells was calculated for the 30-, 40-, and 50-min time points using FlowJo software and is shown next to the profile for each strain. (C) The dia2 mutants exhibit early bud emergence. Cells were treated as in B and budding morphology was assayed by light microscopy. At least 100 cells were counted per time point and the experiment was repeated three times. Representative results from a single experiment are shown. The identity of each strain and bud stage is indicated next to the graph.

The dia2 ΔF-box mutant exhibits growth and S-phase defects. (A) The ΔF-box mutant is cold- and HU-sensitive. The dia2 strain was transformed with DIA2, ΔF-box and empty vector DNA. Serial dilutions of each were then spotted on rich media containing the indicated amount of HU and incubated at either 25 or 30°C. (B) The ΔF-box mutant enters S-phase earlier than WT cells. WT and dia2 ΔF-box cells were arrested in G1 with alpha factor and then released from arrest at 25°C. Samples were taken at the indicated time points and used for flow cytometry. The percentage of S-phase cells was calculated for the 35-, 40-, 45-, and 50-min time points using FlowJo software and is shown next to the profile for each strain. (C) The dia2 ΔF-box mutants exhibit early bud emergence. Cells were treated as in B and budding morphology was assayed by light microscopy. At least 100 cells were counted per time point and the experiment was repeated three times. Representative results from a single experiment are shown. The identity of each strain and bud stage is indicated next to the graph.