Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway - PubMed (original) (raw)

Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway

V Paciotti et al. Mol Cell Biol. 2001 Jun.

Abstract

DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show that mec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Delta cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G(1) or during S phase. Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G(2)/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G(1)/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G(2). The finding that these mutants, although indistinguishable from mec1Delta cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.

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Figures

FIG. 1

mec1 kinase deficiency mutations impair all known DNA damage checkpoints. (A) Serial dilution of cultures of wild-type (wt) YLL334, mec1kd1 sml1Δ DMP2872/8B, mec1kd2 sml1Δ DMP2876/3A, sml1Δ DMP2872/4A, and mec1Δ sml1Δ DMP2882/2C cells growing exponentially in YEPD were spotted on YEPD plates with or without MMS (0.005%) or HU (5 mM). One YEPD plate was UV irradiated (30 J/m2) (UV). (B to E) The strains used were wild-type YLL334, sml1Δ DMP2872/4A, mec1Δ sml1Δ DMP2882/2C, and mec1kd1 sml1Δ DMP2872/8B. (B and C) α-Factor-synchronized cell cultures were UV irradiated (40 J/m2) prior to the release from α-factor in YEPD or were released in YEPD containing 0.02% MMS. (B) Samples of untreated (top), UV-irradiated (middle), or MMS-treated (bottom) cells were taken at the indicated times after release into the cell cycle and analyzed by fluorescence-activated cell sorter. (C) Untreated or UV-irradiated (+UV) cell cultures were scored for the percentage of budded cells at the indicated times. (D) Cell cultures were arrested with nocodazole (noc) and UV irradiated (50 J/m2) prior to the release in YEPD at time zero. Propidium iodide staining was used to directly visualize nuclear division at the indicated times after release from nocodazole in unirradiated and UV-irradiated (+UV) cultures. The survival levels of UV light-treated wild-type, sml1Δ, mec1kd1 sml1Δ, and mec1Δ sml1Δ cells were 78, 90, 8.3, and 7.3%, respectively. (E) Extracts from the above-described G1 UV light-treated (left) or MMS-treated (middle) or G2 UV light-treated (right) cell cultures were analyzed by Western blot assay with anti-Rad53 antibodies. exp, exponentially growing cells.

FIG. 2

Kinase activity and interactions of Mec1kd variants. (A) HA-tagged Mec1 or Mec1kd proteins (Mec1-HA) were immunoprecipitated with anti-HA antibodies (anti-HA IP) from protein extracts prepared from exponentially growing cells concomitantly expressing Mec1-HA9 and Ddc2-MYC18 (DMP3295/8B), Mec1kd1-HA9 and Ddc2-MYC18 (DMP3296/3C), or Mec1kd2-HA9 and Ddc2-MYC18 (DMP3297/6D) from the MEC1 and DDC2 promoters, respectively, as indicated at the bottom. Kinase assays were performed on anti-HA immunoprecipitates, and the results are shown at the top. The same immunoprecipitates were also analyzed by Western blot assay using the antibodies indicated on the right side of the middle and bottom parts of the panel. (B) Immunoprecipitations with anti-HA (anti-HA IP) or anti-MYC (anti-MYC IP) antibodies were performed on extracts from exponentially growing untreated (−) or MMS-treated (+; 0.02% MMS for 1 h) diploid cells with the genotypes indicated in the top part of the panels. Mec1-HA9 and Mec1-MYC18 were then detected by Western blot analysis of the immunoprecipitates by using anti-HA and anti-MYC antibodies. The genotypes of the strains used were MEC1-HA9/MEC1-MYC18 (DMP2750.1), MEC1/MEC1-MYC18 (YLL447.32), MEC1/MEC1-HA9 (YLL476.34), mec1kd1-HA9/MEC1-MYC18 (DMP2885.4), and mec1kd2-HA9/MEC1-MYC18 (DMP2893.1).

FIG. 3

Dominant-negative effect of mec1kd1 overexpression. (A) Serial dilutions of exponentially growing (in YEPD) cultures of wild-type (wt) K699, GAL1-MEC1 YLL516, GAL1-mec1kd1 YLL517, GAL1-mec1kd2 YLL518, and GAL1-mec1kd1 [_GAL-MEC1_] YLL769 cells, all carrying the MEC1 allele at the MEC1 chromosomal locus, and GAL1-mec1kd1 mec1Δ DMP3432/7A and mec1Δ YLL490 cells, both also carrying the sml1Δ allele, were spotted on YEP-raf-gal plates with or without MMS (0.005%) or HU. One YEP-raf-gal plate was UV irradiated (40 J/m2) (UV). (B) Cultures of wild-type DMP3412/1A, GAL1-MEC1 DMP3459/17C, and GAL1-mec1kd1 DMP3455/9A cells, all carrying the MEC1 allele at the MEC1 chromosomal locus, and GAL1-mec1kd1 mec1Δ DMP3432/7A cells, also carrying the sml1Δ allele, logarithmically growing in YEP-raf, were synchronized with α-factor 2.5 h after addition of galactose to 1%. Cell cultures were released from α-factor at time zero into YEP-raf-gal medium with or without 0.02% MMS. One-third of each synchronized culture was UV irradiated (40 J/m2) prior to release. Samples of untreated (top), UV-irradiated (middle), or MMS-treated (bottom) cultures were taken at the indicated times after the release from α-factor and analyzed by fluorescence-activated cell sorter. (C) Cultures of wild-type DMP3412/1A, GAL1-MEC1 DMP3459/17C, GAL1-mec1kd1 DMP3455/9A, GAL1-MEC1 chk1Δ DMP3287/2C, chk1Δ DMP3288/5A, and GAL1-mec1kd1 chk1Δ DMP3288/8C cells, all carrying the MEC1 allele at the MEC1 chromosomal locus, and GAL1-mec1kd1 mec1Δ DMP3432/7A cells, also carrying the sml1Δ allele, logarithmically growing in YEP-raffinose, were synchronized with nocodazole 2 h after addition of 1% galactose and UV irradiated (50 J/m2) prior to release in YEP-raf-gal medium. Nuclear division (top) was directly visualized at the indicated times in untreated and UV light-treated (+UV) cultures by propidium iodide staining. Protein extracts (bottom) from the UV light-treated cell cultures were analyzed by Western blot assay using anti-Rad53 and anti-HA (Chk1) antibodies. exp, exponentially growing cells.

FIG. 4

G1/S and intra-S DNA damage checkpoints in _mec1_-100 and _mec1_-101 mutants. The strains used were wild-type (wt) YLL683.8/4A, mec1Δ sml1Δ DMP3048/5B, _mec1_-100 DMP3343/6C, and _mec1_-101 DMP3344/4A. (A) Serial dilution of exponentially growing (in YEPD) cell cultures were spotted on YEPD plates with or without MMS (0.005%) or HU. One YEPD plate was UV irradiated (40 J/m2) (UV). The data presented in panels B, C, and D all come from the same experiment. (B to D) α-Factor-synchronized cells were released from α-factor at time zero in YEPD (top) or in YEPD containing 0.02% MMS (middle) or were UV irradiated (40 J/m2) prior to the release in YEPD (bottom). (B) Samples of untreated and UV light- and MMS-treated cell cultures were collected at the indicated times after release from α-factor and analyzed by fluorescence-activated cell sorter. (C) Untreated or UV-irradiated (+UV) cell cultures were scored at the indicated times for the percentage of budded cells. (D) Aliquots were removed from the MMS-treated cultures at timed intervals to score for CFU on YEPD plates at 25°C.

FIG. 5

Rad53 and Ddc2 phosphorylation in _mec1_-100 and _mec1_-101 mutants after DNA damage in G1 or during S phase. The strains used were wild-type (wt) YLL683.8/4A, mec1Δ sml1Δ DMP3048/5B, _mec1_-100 DMP3343/6C, and _mec1_-101 DMP3344/4A. The data all come from the experiment described in the legend to Fig. 4B, C, and D. Protein extracts from the UV light-treated (top panel) and the MMS-treated (bottom panel) cell cultures were analyzed by Western blot assay using anti-Rad53 and anti-HA (Ddc2) antibodies. exp, exponentially growing cells.

FIG. 6

G2/M DNA damage checkpoint in _mec1_-100 and _mec1_-101 mutants. Cultures of wild-type (wt) YLL683.8/4A, mec1Δ sml1Δ DMP3048/5B, _mec1_-100 DMP3343/6C, and _mec1_-101 DMP3344/4A cells were arrested with nocodazole and UV irradiated (50 J/m2) prior to release in YEPD. Kinetics of nuclear division were determined as described in the legend to Fig. 1D in untreated and UV light-treated (+UV) cells and are shown at the top. At the bottom is a Western blot analysis of protein extracts from samples of the UV light-treated cell cultures withdrawn at the indicated times. Rad53 and Ddc2 were detected using, respectively, anti-Rad53 and anti-HA (Ddc2) antibodies. exp, exponentially growing cells.

FIG. 7

Response to HU treatment of _mec1_-100 and _mec1_-101 mutants. Cultures of wild-type (wt) YLL683.8/4A, mec1Δ sml1Δ DMP3048/5B, _mec1_-100 DMP3343/6C, and _mec1_-101 DMP3344/4A cells were arrested in G1 with α-factor and then released at time zero in YEPD containing 200 mM HU. Cell samples were collected at the indicated times after the release from α-factor. The data presented in panels A to D all come from the same experiment. (A) DNA content was analyzed by fluorescence-activated cell sorter. (B) Cells were stained with antitubulin antibodies to score for the percentage of cells with elongated spindles by indirect immunofluorescence. (C) Protein extracts were analyzed by Western blot assay using anti-Rad53 antibodies. exp, exponentially growing cells. (D) Appropriate dilutions were plated on YEPD at 25°C to score for CFU

FIG. 8

Amino acid residues changed by the _mec1_-100 mutations. The two Mec1 regions containing the _mec1_-_100_-encoded amino acid changes are shown after alignment of the whole Mec1 amino acid sequence with the S. pombe Rad3 and human ATM and ATR amino acid sequences using the ClustalW program. Identical amino acid residues are shaded in black, and similar residues are highlighted in gray. Residues that are changed in the _mec1_-100 gene product are marked by asterisks.

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Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway - PubMed (original) (raw)