Sensitivity assay to HU and MMS-mediated DNA damage in wild-type, rad53Ha, Δsml1 and rad53Ha Δsml1 mutants. 10-fold serial dilution assays of cultures of the indicated strains exposed to sublethal concentrations of DNA replication inhibitor HU or DNA alkylating chemical MMS.

Forward mutation analysis in wild-type and rad53Ha strains. Canavanine resistance was assayed in rad53Ha and wild-type control cells treated with 0.015% MMS at 28°C for 0, 8 and 24 h. A plot of the resulting forward mutation rate is shown.

A reduction in the levels of the Rad53 checkpoint kinase causes cells to inactivate the checkpoint kinase in response to continuous MMS-induced DNA damage. (A–C) Cultures of wild-type and rad53Ha cells synchronised in G1 with α-factor were released into S-phase in fresh YPAD medium in the presence of the indicated MMS concentrations. Samples were taken at the indicated intervals and prepared for immunoblot and in situ kinase assays (ISA).

The partial inactivation of Rad53 by fusion to a heat-inducible degron renders cells hypersensitive to HU and wild-type-like to MMS. (A), left panels, Western blot analysis of a Rad53-tpd protein in mutant strains asynchronously growing (A) or after partial inactivation of the degron fusion protein at indicated intervals. (A), right panels, comparison of protein levels of Rad53-tpd with appropriate controls (Rad53Ha and wt Rad53). The protein loading controls are portions of respective gels stained with Ponceau. (B) Serial dilutions were made of a Rad53 degron strain (rad53-tpd) together with rad35Ha, Δrad53 Δsml1 and wild-type controls.

Analysis of caffeine effect in rad53Ha resistance to MMS. (A) rad53Ha and wild-type cells were grown at 28°C, blocked in G1 at the same temperature with α-factor for 150 min and released in the presence of 0.010% MMS. Cultures were then divided in two (always in the presence of MMS) either with or without caffeine. (B) The DNA content of cells was determined by flow cytometry at indicated intervals of the experiment described in (A). (C) Cell viability during the course of the experiment.

Limited RNR2 expression in rad53Ha mutant cells in response to blocks in DNA replication and DNA damage. Northern blot analyses of RNA isolated from wild-type and rad53Ha cells treated with HU (200 mM) (A) or MMS (0.033%) (B). Wild-type and Rad53Ha-tagged cells were synchronised with α-factor and then released in the presence of HU or MMS. RNA samples were isolated at the times indicated and then electrophoresed, blotted to nylon membranes and the membranes were probed for RNR2 expression. The loading control is the 18S rRNA stained with methylene blue.

Recovery from replication arrest (HU) and DNA damage (MMS) in rad53Ha cells. Wild-type and rad53Ha mutant cells growing exponentially at 28°C were first synchronised in G1 with α-factor and then released into S-phase in fresh YPAD medium in the presence of HU (A) or MMS (B) and incubated for a further 90 min. Cultures were then released from drug exposure into fresh YPAD medium and further incubated at 28°C (recovery). Protein extracts were prepared at the indicated intervals and analysed by western blotting using an anti-Rad53 polyclonal antibody and in situ kinase assay (ISA).

Effect of HU treatment on initiation from an early (ARS306) origin of DNA replication in rad53Ha cells. (A) wild-type and rad53Ha mutant cells were grown in YPAD medium to exponential phase, synchronised with α-factor in G1 and then released into fresh YPAD medium containing 0.2 M HU. Genomic DNA was prepared from cells at indicated intervals (from the release) and cut with NcoI. Restriction fragments were electrophoresed in N:N 2D-gels, transferred to nylon membranes and hybridized to a probe spanning the ARS306 early origin of replication. (B) rad53Ha mutant cells acumulate abnormal DNA replication structures (cone-shaped signals and small Ys as indicated by arrows). A drawing of the abnormal intermediates related to DNA replication fork collapse, according to Lopes et al. (2001), is also shown.

Suppression of MMS-induced DNA damage and HU-replicative stress sensitivity defects by increased expression of the rad53Ha allele. (A) 10-fold serial dilution assay of indicated strains. Cells growing in YPAD medium were spotted onto YPAD (2% glucose, labeled as GAL1 OFF) or YPARG (0.3% galactose, 1% raffinose, labeled as GAL1 ON). Petri dishes with no drug (control), with 10 mM HU or with 0.015% MMS (as indicated), were incubated for 48 h at 28°C and then photographed. (B) Analysis of Rad53 protein levels (Immunoblot) and Rad53 activity (ISA) in asynchronous (control) HU- and MMS-treated cultures of wild-type (1), rad53Ha (2) and GAL1,10:rad53Ha sml1Δ (3) strains. When treated, yeast cultures were exposed to HU or MMS (as indicated) for 90 min.

The Ha carboxy-terminal tagging of Rad53 reduces protein levels and renders cells sensitive to HU and resistant to MMS. (A) 10-fold serial dilutions of cultures of wild-type and rad53Ha mutant cells growing in YPAD and spotted onto YPAD plates with or without HU (50 mM) or MMS (0.01%) as indicated. Note that the assay was performed in the 15Dau and W303 S.cerevisiae strain backgrounds. (B) Immunoblot analysis and In situ Kinase Assay of Rad53 in asynchronous cultures of wild-type and rad53Ha strains (A), or in cells treated for 90 min with 200 mM HU (H), or with 0.033% MMS (M), or with 10 mU/ml of Bleomycin (B) (Note: a longer exposure of the western and ISA were needed to detect Rad53Ha in bleomycin samples). (C) Cultures of wild-type and rad53Ha strains growing exponentially in YPAD medium were synchronised in G1 with alpha-factor pheromone at 28°C and then released into fresh YPAD medium at the same temperature to allow cells to initiate S-phase synchronously. Samples were taken at indicated intervals and processed for viability assays and percentage of viables was plotted.

MMS-induced S-phase DNA damage checkpoint in rad53Ha mutant cells. Cultures of wild-type and rad53Ha mutant cells presynchronised in G1 with α-factor were released into fresh YPAD medium containing 0.033% MMS to induce the intra S-phase DNA damage checkpoint response. Samples were taken at the indicated intervals and processed for FACS analysis, 2D-gel analysis of ARS306 and ARS501, Western blotting and ISA assays of Rad53. (A) Genomic DNA samples were prepared from aliquots at the indicated intervals and cut with NcoI, restriction fragments were electrophoresed in 2D-gels, transferred to nylon membranes and hybridized to probes spanning the ARS306 and ARS501 origins of DNA replication. (B) FACS analysis of the DNA content of rad53Ha and wild-type control cells in response to MMS treatment. Note that rad53Ha mutants passed through S-phase faster than wt controls (samples indicated by arrows). (C) Protein extracts from aliquots from the same samples were analysed by Western blot assays with anti-Rad53 antibody or In Situ Authophosphorylation assays (ISA), as indicated, to test the activation of Rad53. A cross-referenced sample (labelled C) was used in both the Western and ISA assays that corresponded to the 120 min sample of rad53Ha or wild-type experiments respectively. (D) Genomic DNA samples from Δrad53 Δsml1 cells treated with MMS were prepared at indicated intervals as in section A and hybridized to the ARS306 probe. Note that replication in these Δrad53 Δsml1 cells starts 15 min later than in wild-type and rad53Ha cells in A, and also that small Ys and cone-shaped signals are evident from 45 min (to the end of the experiment) indicating genuine DNA replication fork collapse. (E) FACS analysis of Δrad53 Δsml1 cells in D treated with MMS. (F) Comparison of replication intermediates in rad53HA and Δrad53 Δsml1 cells (cone-shaped signals and small Ys are indicated by arrows in the Δrad53 Δsml1 mutant).