Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Abstract

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.

FIG. 1.

Arrest of cell cycle progression by a single unrepaired DSB in wild-type and mutant cells. (A) Arrest and adaptation of cells experiencing an irreparable DSB in G₁. At least 300 G₁ unbudded cells were initially plated (gray bar). The numbers of cells and buds at 8 h (hatched bar) and 24 h (black) are shown. After 24 h, wild-type cells initially plated as G₁ unbudded cells onto galactose-containing medium to induce HO endonuclease have mostly adapted and resumed cell division, whereas adaptation-defective tid1Δ and rad51Δ strains remain arrested prior to anaphase. (B) Results of FACS analysis of wild-type and mutant cells induced for HO expression at 0 h. The apparent increase in DNA content of checkpoint-arrested cells above the 2C level is caused by light scattering of G₂/M-arrested cells because of their greatly enlarged size and is not an indication of continued DNA replication (53).

FIG. 2.

Adaptation of wild-type and mutant cells. Cells progressing beyond the two-cell-plus-bud stage at 24 h after HO induction were scored as having adapted. (A) Effect of rad9Δ on the arrest and adaptation of wild-type and mutant cells at 8 and 24 h. Also shown are the effects of rfa1-t11 (B), mre11Δ (C), and rad52Δ (D) on the adaptation of wild-type and mutant cells at 24 h.

FIG. 3.

5′-to-3′ resection of an HO-induced DSB in adaptation-defective mutants. (A) Slot blot hybridization was performed with a probe specific for the strand ending 5′ distal to the HO-cleaved MAT locus. (B) Percentages of the sequences remaining at different times. wt, wild type.

FIG. 4.

Lack of binding of Rad51-K191A protein to ssDNA in vivo. Rad51p binding to ssDNA adjacent to a HO endonuclease-induced DSB was monitored by chromatin immunoprecipitation (IP) in a strain that cannot repair the break by homologous recombination. The amount of input DNA was monitored by PCR amplification of the arg5,6 gene, and the same primer pairs were used to show the low background of arg5,6 DNA in the samples immunoprecipitated by antibody against Rad51.

FIG. 5.

Phosphorylation and kinase activity of Rad53p. The activation of Rad53p kinase is shown both by Western blot analysis (top of each set), in which phosphorylated forms of Rad53p exhibit slower migration, and by an in-gel activity autophosphorylation assay. (A) Activation of Rad53p by a single HO-induced DSB in logarithmically growing wild-type and adaptation-defective mutant cells. L represents cells in logarithmic growth prior to HO induction. (B) Suppression of the adaptation defect of rad51Δ cells by the rfa1-t11 mutation. (C) Persistent hyperphosphorylation of Rad53p in rad52Δ rfa1-t11 cells after induction of an irreparable DSB. This is not seen for either rad52Δ or rfa1-t11 alone. No autophosphorylation assay was performed.

FIG. 6.

A C-terminal truncation of Rad52 (Δ409-420) is adaptation defective. A rad52Δ rfa1-t11 strain was transformed with a centromeric plasmid carrying either RAD52 (B) or rad52Δ409-420 (C) or with an empty vector (A). G₁ cells were plated onto YEP-galactose as described above. Cells were scored at 8 h (hatched bars) and 24 h (black bars) after induction of the DSB.