Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response

Abstract

The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.

Figure 1

HU induces Rfa1p phosphorylation. (A) Western blot analysis of Rfa1p from TWY397 cells harvested immediately before and after HU exposure. (B) Western blot analysis of Rfa1p from native extract of HU-treated TWY397 cells treated with λPPase and sodium orthovanadate as indicated. For both experiments, TWY397 cells were incubated at 30°C.

Figure 2

Mec1p and Tel1p are required for HU-induced Rfa1p phosphorylation. (A) Western blot analysis of Rfa1p from exponentially growing (–) or HU-treated (+) cells. Strains employed were: TWY397 (wt), TWY308 (mec1), TWY312 [rad53 (= mec2)], TWY316 (mec3) and TWY398 (rad9). P-Rfa1p, phosphorylated form of Rfa1p. (B) YDM937 (mec1-1 tel1Δ1) cells transformed with pRS316 (vector), pRS316 containing TEL1 (pTEL1) or pRS316 containing MEC1 (pMEC1) were analyzed as in (A). *, immunoreactive band that migrates faster than full-length Rfa1p and is presumed to be an Rfa1p degradation product. The intensity of this band varies between experiments.

Figure 3

Radiation induces checkpoint-dependent Rfa1p phosphorylation. Wild-type and checkpoint mutant cells were untreated (–) or challenged with UV radiation (+; 60 J/m²). Rfa1p in denatured extracts of these cells was detected by western blot analysis. The strains employed were the same as those indicated in Figure 2A, except DLY285 (MATa mec1-1) was used instead of TWY308 (MATα mec1-1).

Figure 4

RPA phosphorylation induced by telomeric damage is dependent on Mec1p. Western blot analysis of Rfa1p and Rfa2p from cdc13 cells harvested immediately before and after incubation at the restrictive temperature for 2 h. Strains employed were: TWY146 (cdc13), TWY158 (cdc13 mec1-1), TWY149 (cdc13 mec1-2) and TWY150 (cdc13 mec1-3). P-Rfa2p, phosphorylated form of Rfa2p.

Figure 5

Rfa1p and Rfa2p phosphorylation induced by telomeric damage exhibit different genetic dependencies. (A) Western blot analysis of RPA from cells harvested immediately before and after incubation at the restrictive temperature for 2 h. Strains employed were DLY408 (cdc13), DLY409 (cdc13 rad9), DLY410 (cdc13 rad24), DLY411 (cdc13 rad9 rad24), DLY418 (- - -) and DLY419 (rad9). These strains contain a mutant allele of CDC15 that allows for mitotic arrest of checkpoint-deficient strains at the restrictive temperature (40). (B) Western blot analysis of Rfa2p from DLY409 cells incubated at the restrictive temperature for 1 h. (C) Phosphatase treatment of native extract from DLY409 cells incubated at the restrictive temperature for 2 h.

Figure 6

DNA-PKcs catalyzes phosphorylation of yeast RPA. Human and yeast RPA were compared as substrates of DNA-PKcs employing a radioassay with purified proteins (see Materials and Methods). Bands corresponding to phosphorylated RPA are indicated, as well as the approximate positions of the unphosphorylated subunits. h, human; y, yeast; RPA1, human RPA large subunit; RPA2, human RPA middle subunit.