DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient Saccharomyces cerevisiae

Abstract

In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair. We have established in yeast a connection between Ku activity and DNA double-strand-break damage repair, and a connection between Ku activity and commitment to DNA replication. We generated double-stranded DNA breaks in yeast cells in vivo by expressing a restriction endonuclease and have shown that yeast mutants lacking Ku p70 activity died while isogenic wild-type cells did not. Moreover, we have discovered that DNA damage occurs spontaneously during normal yeast mitotic growth, and that Ku functions in repair of this damage. We also observed that mitotically growing Ku p70 mutants have an anomalously high DNA content, suggesting a role for Ku in regulation of DNA synthesis. Finally, we present evidence that Ku p70 function is conserved between yeast, Drosophila, and humans.

Figure 1

Replica plating experiment showing the phenotypic lag of the temperature-sensitive growth of the hdf1 null strain, BRY1, at 37°C. BRY1 was transformed with (A) the vector (pCH37), (B) the HDF1 gene on a plasmid, or (C) the pCH37/IRBP (Drosophila Ku p70 cDNA) plasmid. Transformants were selected on medium containing glucose and then streaked to medium containing galactose and incubated at 37°C (plate on left). After 3 days, the plate was replica plated to a fresh galactose plate and the second plate was incubated at 37°C (plate on right).

Figure 2

Immunofluorescence analysis of the hdf1Δ null (BRY2) cells incubated at 37°C for 8 h. (Upper) Antitubulin staining. (Lower) The same cells with DAPI staining to localize the nucleus. (Bar = 5 μm.)

Figure 3

Relative transcriptional activity in hdf1Δ null and wild-type strains as monitored by GFP activity. GFP activity expressed in yeast by seven different promoter fusion constructions. The promoter tested in each case is listed at the base of a pair of GFP levels. GFP levels have been normalized to the wild-type level for each promoter fusion construction. GFP levels in mutant hdf1Δ null cells (BRY2) are shown in striped boxes, and GFP levels in wild-type cells (BRY3) are shown in solid boxes. Identical results were obtained at 20°C and 37°C. Standard deviation for each data set was 10% or less.

Figure 4

FACS analysis of (A) wild-type cells (BRY3) growing exponentially at 20°C, (B) hdf1Δ null cells (BRY2) growing exponentially at 20°C, (C) hdf1Δ null cells shifted to 37°C and incubated for 3 h, and (D) hdf1Δ null cells shifted to 37°C and incubated for 22 h. The y-axis shows relative number of cells, and the x-axis shows relative DNA content.

Figure 5

Effect of α-factor on wild-type and Ku p70-deficient yeast. FACS profiles of wild-type and hdf1Δ null cells were incubated at 20°C before (A and C) and after (B and D) 6-h treatment with α-factor. Wild-type profiles are shown on the left (A and B); hdf1Δ null profiles on right (C and D). B and D show the 6-h α-factor arrest FACS profile in black superimposed on the FACS profiles from A and C, respectively, in gray.

Figure 6

EcoRI endonuclease expression experiment. All strains were grown at 20°C on medium containing glucose and then streaked onto medium containing galactose and incubated at 20°C. (A) hdf1Δ null strain (BRY1) transformed with the EcoRI-producing plasmid YCpGalRIb. (B) hdf1Δ null strain (BRY1) transformed with the control vector. (C) Isogenic wild-type cells (BRY4) transformed with the EcoRI plasmid, YCpGalRIb.

Figure 7

Complementation of the hdf1Δ null strain temperature-sensitive growth defect. (A) hdf1Δ null strain (BRY1) transformed with the control vector pCH37. (B) BRY1 transformed with pCH37/IRBP. (C) BRY1 transformed with pCH37/HuKu. Cells were grown at 20°C and then streaked onto medium containing galactose and incubated at 37°C.