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, 23 (2), 429-38

Physical and Functional Interactions Between Nucleotide Excision Repair and DNA Damage Checkpoint

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Physical and Functional Interactions Between Nucleotide Excision Repair and DNA Damage Checkpoint

Michele Giannattasio et al. EMBO J.

Abstract

The mechanisms used by checkpoints to identify DNA lesions are poorly understood and may involve the function of repair proteins. Looking for mutants specifically defective in activating the checkpoint following UV lesions, but proficient in the response to methyl methane sulfonate and double-strand breaks, we isolated cdu1-1, which is allelic to RAD14, the homolog of human XPA, involved in lesion recognition during nucleotide excision repair (NER). Rad14 was also isolated as a partner of the Ddc1 checkpoint protein in a two-hybrid screening, and physical interaction was proven by co-immunoprecipitation. We show that lesion recognition is not sufficient for checkpoint activation, but processing, carried out by repair factors, is required for recruiting checkpoint proteins to damaged DNA. Mutations affecting the core NER machinery abolish G1 and G2 checkpoint responses to UV, preventing activation of the Mec1 kinase and its binding to chromosomes. Conversely, elimination of transcription-coupled or global genome repair alone does not affect checkpoints, suggesting a possible interpretation for the heterogeneity in cancer susceptibility observed in different NER syndrome patients.

Figures

Figure 1
Figure 1
cdu1-1 is UV sensitive and shows a lesion-specific Rad53 phosphorylation defect. Strains were wt (K699), mec1-1 (DMP2697/2c) and cdu1-1 (derived from K699). (A) Serial dilutions of exponential cultures were spotted onto YEPD plates and mock treated or UV irradiated (50 J/m2); a third plate contained 0.02% MMS. The photograph was taken after 2 days of incubation. (B) Exponential cultures were treated with 0.02% MMS (bottom panel) or arrested in G1 with α-factor and in G2 with nocodazole. In panel B (top), blocked cells were either irradiated with UV (50 J/m2) or treated with 4NQO (2 μg/ml). (C) Zeocin was added at the indicated concentrations. Checkpoint activation was monitored by evaluating Rad53 phosphorylation in Western blotting.
Figure 2
Figure 2
cdu1-1 exhibits defective G1/S and G2/M cell cycle arrests after UV irradiation. Strains were as in Figure 1 and mec3Δ (YMIC4E6). Exponential cultures were arrested in G1 (A) or G2 (B) and UV irradiated (40 J/m2). The cells were then allowed to progress through the cell cycle. G1/S and G2/M checkpoint arrests were monitored by evaluating, at the indicated times, the percentage of budded cells (A) or uninucleated cells (B), respectively.
Figure 3
Figure 3
Inactivation of genes involved in the early steps of NER prevents Mec1-dependent phosphorylation of Rad9 and Mec1 kinase activation, which requires processing of the primary lesion. (A) Strains were wt (YMIC5B5), rad14Δ (YMIC8B3) and rad2Δ (YMIC8C2). Exponential cells were arrested in G1 and G2 and treated with 4NQO. TCA extracts were analyzed by Western blotting with antibodies against the MYC tag or Rad53, in order to evaluate Rad9 and Rad53 phosphorylation, respectively. The * and ** symbols indicate the cell cycle-dependent phosphorylation of Rad9, normally observed in G2, and the damage-dependent hyperphosphorylation, respectively. (B) Strains were wt (YLL683.8/3b), rad14Δ (YMIC8B9), rad2Δ (YMIC8B7) and rad3K48R (YMIC12D3). Cells were blocked in G2 with nocodazole and treated with 4NQO (2 μg/ml) or zeocin (200 μg/ml). Ddc2 and Rad53 phosphorylation was assayed by Western blotting with anti-HA tag or anti-Rad53 antibodies, after TCA protein extraction. Similar results were obtained with UV irradiation (not shown).
Figure 4
Figure 4
Either GGR or TCR alone is sufficient to trigger properly the checkpoint response following UV radiation. Strains were wt (K699), rad7Δ (YMIC12I1), rad4Δ (YMIC12H6), rad2Δ (YMIC8B7), rad14Δ (YMIC12H6), rad3K48R (YMIC10H2), rad26Δ (YMG30) and rad7Δrad26Δ (YMG48/9d). Exponential cultures were arrested in G1 and G2 and mock or UV (100 J/m2) treated. Rad53 phosphorylation was analyzed by Western blotting. Rad53 kinase activity was monitored by in situ kinase assay, as previously described (Pellicioli et al, 1999).
Figure 5
Figure 5
Rad14 physically interacts with the PCNA-like complex. (A) EGY48 cells containing the Rad14 prey plasmid (pJG4-5 Rad14TH1) were transformed with the plasmids expressing the indicated baits. Cells were spotted on plates (SC-HIS, TRP, URA) containing (+LEU) or lacking (−LEU) leucine to select for interactors. The same cells were also spotted on SC-HIS, TRP, URA plates, containing XGAL to monitor lacZ reporter expression. The sugar source was as indicated (Glu: 2% glucose, to shut off prey expression; Gal: 2% raffinose, 2% galactose, to turn on prey expression). + indicates the positive control (p53 bait, SV40 Tag prey). (B) myc-tagged Rad14 was immunopurified, using anti-myc antibodies crosslinked to protein G–sepharose, from Rad14-Myc or Rad14 cells treated or mock treated with 4NQO. The recovered samples were separated by SDS–PAGE and analyzed by Western blotting with Ddc1-specific antibodies. Control lanes (Ddc1 control and Ddc1Δ control) contain TCA extracts from wt and ddc1Δ cells, respectively. Strains were RAD14 (K699), RAD14-myc (YMIC7E5) and ddc1Δ (YLL244). (C) The GST fusion proteins were purified from E. coli cells and incubated with 35S-labeled in vitro-translated Rad14. Proteins bound to the fusions were recovered, separated by SDS–PAGE and analyzed by autoradiography.
Figure 6
Figure 6
NER is required for loading Mec1–Ddc2 kinase and Ddc1 complexes onto damaged chromosomes. (A) Strains were wt (YLL683.8/3b) and rad14Δ (YMIC8B9). Cultures were arrested in G1 and treated with 4NQO or zeocine. Cells were lysed and processed as described in Materials and methods, and proteins associated with bulk chromatin were analyzed by SDS–PAGE and Western blotting with anti-HA tag antibodies. (B) All strains, except for untagged controls, contained either DDC1-27MYC or DDC2-18MYC, as indicated. Chromosome spreads were prepared from G1 cells that had been mock treated or UV treated (100 J/m2), as described in Materials and methods. Samples were examined by indirect immunofluorescence and by DAPI staining. Similar experiments performed with G2 cells gave similar results. Strains were DDC1-MYC (YLL444.9), DDC2-MYC (YLL733.1), DDC1-MYC rad14Δ (YMG44), DDC2-MYC rad14Δ (YMG39), DDC1-MYC rad3K48R (YMG36), DDC2-MYC rad3K48R (YMG38), DDC1 (K699) and DDC2 (K699).

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