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
rad51
Δ.
rad52
Δ also suppresses adaptation defects of
srs2
Δ mutants but not those of
yku70
Δ or
tid1
Δ mutants. Neither
rad54
Δ nor
rad55
Δ 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
rad51
Δ mutants and fully restores adaptation in
yku70
Δ and
tid1
Δ mutants. Surprisingly, although neither
rfa1-t11
nor
rad52
Δ mutants are adaptation defective, the
rad52
Δ
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.