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Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1.

Penedos A, Johnson AL, Strong E, Goldman AS, Carballo JA, Cha RS - PLoS ONE (2015)

Bottom Line: A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases.In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest.Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, London, NW7 1AA, United Kingdom.

ABSTRACT
A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.

No MeSH data available.


Related in: MedlinePlus

Lack of the Hop1-phospho-S298 leads to temperature- and dose- dependent meiotic failure.(A) Schematic representation of Hop1 with the locations of eight [S/T]Q motifs. S: serine, T: threonine, SCD: [S/T]Q Cluster Domain. Also shown are the HORMA domain, Zn finger motif, and nuclear localization signal (NLS). (B) and (C) Specificity of the phospho-specific α-pS298 and α-pT318 antibodies. Nuclear spreads of HOP1 and hop1-S298A panel (B) or HOP1 and hop1-T318A panel (C) were prepared from samples taken at 5hours after induction of synchronous meiosis at 23°C. The spreads were stained with DAPI and the antibodies against either the phospho-S298 panel (B) or the phospho-T318 panel (C). (D) and (E). In vivo phosphorylation of Hop1-S298 and T318 during DMC1 (D) or dmc1Δ (E) meiosis at 23°C. Nuclear spreads of a DMC1 or dmc1Δ strain were prepared from samples collected at the indicated time points. The spreads were stained with the antibodies against Hop1, HA (for detection of Mek1-HA), and the two phospho-specific antibodies, α-pS298 and α-pT318. A nucleus exhibiting 10 or more foci of each epitope was scored as a positive. Also shown are the fractions of cells having undergone first meiotic division or meiosis I (MI) at each time point. Errors were calculated from the 95% confidence interval of a binomial distribution. (F) Spore viability of homozygous diploid strains of indicated genotype at specified temperature. For each genotype, at least 80 spores were analysed. A: Alanine; D: aspartic acid, hop1-S298Ax2: an allele containing two tandem copies of hop1-S298A. hop1-SCD: an allele where the S298, S311, and T318 in the SCD are mutated to A [6]. hop1-S311A: an allele where the S311 is mutated to A. (G) Spore viability of indicated HOP1 alleles at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1). (H) Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background. (I). Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1).
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pone.0134297.g001: Lack of the Hop1-phospho-S298 leads to temperature- and dose- dependent meiotic failure.(A) Schematic representation of Hop1 with the locations of eight [S/T]Q motifs. S: serine, T: threonine, SCD: [S/T]Q Cluster Domain. Also shown are the HORMA domain, Zn finger motif, and nuclear localization signal (NLS). (B) and (C) Specificity of the phospho-specific α-pS298 and α-pT318 antibodies. Nuclear spreads of HOP1 and hop1-S298A panel (B) or HOP1 and hop1-T318A panel (C) were prepared from samples taken at 5hours after induction of synchronous meiosis at 23°C. The spreads were stained with DAPI and the antibodies against either the phospho-S298 panel (B) or the phospho-T318 panel (C). (D) and (E). In vivo phosphorylation of Hop1-S298 and T318 during DMC1 (D) or dmc1Δ (E) meiosis at 23°C. Nuclear spreads of a DMC1 or dmc1Δ strain were prepared from samples collected at the indicated time points. The spreads were stained with the antibodies against Hop1, HA (for detection of Mek1-HA), and the two phospho-specific antibodies, α-pS298 and α-pT318. A nucleus exhibiting 10 or more foci of each epitope was scored as a positive. Also shown are the fractions of cells having undergone first meiotic division or meiosis I (MI) at each time point. Errors were calculated from the 95% confidence interval of a binomial distribution. (F) Spore viability of homozygous diploid strains of indicated genotype at specified temperature. For each genotype, at least 80 spores were analysed. A: Alanine; D: aspartic acid, hop1-S298Ax2: an allele containing two tandem copies of hop1-S298A. hop1-SCD: an allele where the S298, S311, and T318 in the SCD are mutated to A [6]. hop1-S311A: an allele where the S311 is mutated to A. (G) Spore viability of indicated HOP1 alleles at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1). (H) Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background. (I). Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1).

Mentions: Hop1 contains eight ATM/ATR consensus sites (nine in the SK1 strain background), referred to as SQ/TQ motifs, each comprising of a serine (S) or threonine (T) followed by a glutamine (Q) (Fig 1A). Of the eight SQ/TQ motifs, the phospho-T318 is required for the essential recruitment and activation of Mek1, while the threonine at position 181 might play a different role [6]. When replacing any of the remaining SQ/TQ sites to alanine, a residue that cannot be phosphorylated, all of the mutant alleles appear to behave indistinguishably from the wild type during unchallenged meiosis, except for the serine 298 (S298), elimination of which confers a modest reduction in spore viability [6] (below).


Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1.

Penedos A, Johnson AL, Strong E, Goldman AS, Carballo JA, Cha RS - PLoS ONE (2015)

Lack of the Hop1-phospho-S298 leads to temperature- and dose- dependent meiotic failure.(A) Schematic representation of Hop1 with the locations of eight [S/T]Q motifs. S: serine, T: threonine, SCD: [S/T]Q Cluster Domain. Also shown are the HORMA domain, Zn finger motif, and nuclear localization signal (NLS). (B) and (C) Specificity of the phospho-specific α-pS298 and α-pT318 antibodies. Nuclear spreads of HOP1 and hop1-S298A panel (B) or HOP1 and hop1-T318A panel (C) were prepared from samples taken at 5hours after induction of synchronous meiosis at 23°C. The spreads were stained with DAPI and the antibodies against either the phospho-S298 panel (B) or the phospho-T318 panel (C). (D) and (E). In vivo phosphorylation of Hop1-S298 and T318 during DMC1 (D) or dmc1Δ (E) meiosis at 23°C. Nuclear spreads of a DMC1 or dmc1Δ strain were prepared from samples collected at the indicated time points. The spreads were stained with the antibodies against Hop1, HA (for detection of Mek1-HA), and the two phospho-specific antibodies, α-pS298 and α-pT318. A nucleus exhibiting 10 or more foci of each epitope was scored as a positive. Also shown are the fractions of cells having undergone first meiotic division or meiosis I (MI) at each time point. Errors were calculated from the 95% confidence interval of a binomial distribution. (F) Spore viability of homozygous diploid strains of indicated genotype at specified temperature. For each genotype, at least 80 spores were analysed. A: Alanine; D: aspartic acid, hop1-S298Ax2: an allele containing two tandem copies of hop1-S298A. hop1-SCD: an allele where the S298, S311, and T318 in the SCD are mutated to A [6]. hop1-S311A: an allele where the S311 is mutated to A. (G) Spore viability of indicated HOP1 alleles at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1). (H) Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background. (I). Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1).
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pone.0134297.g001: Lack of the Hop1-phospho-S298 leads to temperature- and dose- dependent meiotic failure.(A) Schematic representation of Hop1 with the locations of eight [S/T]Q motifs. S: serine, T: threonine, SCD: [S/T]Q Cluster Domain. Also shown are the HORMA domain, Zn finger motif, and nuclear localization signal (NLS). (B) and (C) Specificity of the phospho-specific α-pS298 and α-pT318 antibodies. Nuclear spreads of HOP1 and hop1-S298A panel (B) or HOP1 and hop1-T318A panel (C) were prepared from samples taken at 5hours after induction of synchronous meiosis at 23°C. The spreads were stained with DAPI and the antibodies against either the phospho-S298 panel (B) or the phospho-T318 panel (C). (D) and (E). In vivo phosphorylation of Hop1-S298 and T318 during DMC1 (D) or dmc1Δ (E) meiosis at 23°C. Nuclear spreads of a DMC1 or dmc1Δ strain were prepared from samples collected at the indicated time points. The spreads were stained with the antibodies against Hop1, HA (for detection of Mek1-HA), and the two phospho-specific antibodies, α-pS298 and α-pT318. A nucleus exhibiting 10 or more foci of each epitope was scored as a positive. Also shown are the fractions of cells having undergone first meiotic division or meiosis I (MI) at each time point. Errors were calculated from the 95% confidence interval of a binomial distribution. (F) Spore viability of homozygous diploid strains of indicated genotype at specified temperature. For each genotype, at least 80 spores were analysed. A: Alanine; D: aspartic acid, hop1-S298Ax2: an allele containing two tandem copies of hop1-S298A. hop1-SCD: an allele where the S298, S311, and T318 in the SCD are mutated to A [6]. hop1-S311A: an allele where the S311 is mutated to A. (G) Spore viability of indicated HOP1 alleles at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1). (H) Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background. (I). Sporulation efficiency of dmc1Δ strains in the indicated hop1 mutation background at 23°C as a either homozygous diploid (allele/allele), hemizygous diploid (allele/ hop1Δ) or heterozygous diploid (allele/HOP1).
Mentions: Hop1 contains eight ATM/ATR consensus sites (nine in the SK1 strain background), referred to as SQ/TQ motifs, each comprising of a serine (S) or threonine (T) followed by a glutamine (Q) (Fig 1A). Of the eight SQ/TQ motifs, the phospho-T318 is required for the essential recruitment and activation of Mek1, while the threonine at position 181 might play a different role [6]. When replacing any of the remaining SQ/TQ sites to alanine, a residue that cannot be phosphorylated, all of the mutant alleles appear to behave indistinguishably from the wild type during unchallenged meiosis, except for the serine 298 (S298), elimination of which confers a modest reduction in spore viability [6] (below).

Bottom Line: A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases.In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest.Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, London, NW7 1AA, United Kingdom.

ABSTRACT
A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis.

No MeSH data available.


Related in: MedlinePlus