Pre-activation of the genome integrity checkpoint increases DNA damage tolerance

Abstract

The genome integrity checkpoint is a conserved signaling pathway that is regulated in yeast by the Mec1 (homologous to human ATR) and Rad53 (homologous to human Chk1) kinases. The pathway coordinates a multifaceted response that allows cells to cope with DNA damage and DNA replication stress. The full activation of the checkpoint blocks origin firing, stabilizes replication forks, activates DNA repair proteins and may lead to senescence or apoptosisin higher eukaryotes. We have recently demonstrated that endogenous replication stress can activate the genome integrity checkpoint in budding yeast at a low level that does not go so far as to interfere with cell cycle progression, but it does activate DNA damage-inducible proteins. Here we demonstrate that the low level pre-activation of the checkpoint, either by endogenous replication stress or by the nucleotide-depleting drug hydroxyurea, can increase damage tolerance to multiple DNA-damaging agents. These results may provide new strategies for using the checkpoint to protect normal cells from genotoxic stress.

Figure 1.

Deletion of IXR1 increases DNA damage tolerance. (A) Spot assays on YPD media with various drugs were performed using 1:10 serial dilutions (1:5 for MMS) of logarithmically growing cultures of wild-type (W1588-4C), ixr1Δ (TOY736), ixr1Δ smlΔ (TOY714), ixr1Δ dun1Δ sml1Δ (TOY730), sml1Δ (U952-3B), dun1Δ (TOY510) and dun1Δ sml1Δ (TOY728) strains. (B) Analysis of DNA damage tolerance. Wild-type (W1588-4C, open circles) and ixr1Δ (TOY736, black triangles) cells were grown to mid-log phase, and appropriate dilutions were plated on YPD containing the indicated amounts of various drugs or on YPD alone and were UV irradiated at the indicated doses. (C) Spot assays were performed as in Figure 1A using following strains: wild-type (W1588-4C), ixr1Δ (TOY736), ixr1Δ smlΔ (TOY714), ixr1Δ mec1Δ sml1Δ (TOY753) and mec1Δ sml1Δ (TOY757).

Figure 2.

The genome integrity checkpoint is pre-activated in ixr1 mutants and is activated further in response to DNA-damaging drugs and replication block. (A) Western blot analysis of Rnr3 and Rnr4 levels in wild-type (wt) (TOY632) and ixr1Δ (TOY732) strains before and after 2-h treatments with 0.2 mg/l 4-NQO, 0.02% MMS, 200 mM HU, 2.5 mMcisplatin or 325 μM t-BHP. Protein levels were quantified in relative units (RU, levels of Rnr3 or Rnr4 divided by the level of tubulin in the same sample lane as described in ‘Materials and Methods’ section). ND: not detected. (B) Western blot analysis of Rnr3, Rnr4 and Sml1 levels in wild-type (wt) (TOY632) and ixr1Δ (TOY732) strains. Cells were synchronized with α-factor, released into fresh YPD media and collected at the indicated time points. Upper panel: corresponding flow cytometer charts. as: asynchronous culture. RU, relative units; ND, not detected.

Figure 3.

Low concentration of HU improves DNA damage tolerance of the wild-type strain. Western blot analysis of Rnr1–4 protein levels and corresponding flow cytometry histograms (A) and dNTP concentration measurement (B) in the wild-type strain (W1588-4C) after 10 h incubation with 0, 5, 12, 15 or 20 mM HU. (C–E) Analysis of DNA damage tolerance of the wild-type strain incubated 10 h with 0, 5, 12, 15 or 20 mM HU. Cells were spun, washed once with water, and appropriate dilutions were plated on YPD or YPD containing 0.15 mg/l 4-NQO (C), 2 mMcisplatin (D) or 325 μM t-BHP (E). (F) Spot assays on YPD media containing 0.15 mg/l or 0.17 mg/l 4-NQO were performed using 1:10 serial dilutions of wild-type cells pre-incubated 10 h with 0 mM HU (top row) or 12 mM HU (bottom row). (G) Analysis of DNA damage tolerance. Wild-type cells (W1588-4C) were grown to mid-log phase and appropriate dilutions were plated on YPD containing 325 μM t-BHP with or without 15 mM HU. The number of colonies is shown.