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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

Hop1-S298 phosphorylation is required for dmc1Δ-dependent meiotic checkpoint response.(A) and (B). Fraction of cells undergone meiosis I (MI) in synchronous meiotic cultures of HOP1, hop1-S298A, and hop1-T318A at 23°C in a DMC1 or dmc1Δ background. T50: Time at which 50% of the active culture has completed MI. At least three separate timecourses were considered, for each set of strains for each background. T50 kinetics were calculated from the most representative timecourse for each set of strain and DMC1/dmc1 background. Errors were calculated from the 95% confidence interval of a binomial distribution. (C) and (D). Effects of hop1-S298A and hop1-T318A on Mek1-HA phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-HA antibody. Positions of the unphosphorylated and phosphorylated Mek1-HA species are indicated to the right of the blot. ‘% pMek’ corresponds to the proportion of phosphorylated Mek1-HA species in each lane calculated by dividing the signal found in the ‘pMek1-HA’ area of the gel by the total signal (‘pMek1-HA’ + ‘Mek1-HA’). (E) and (F). Effects of hop1-S298A and hop1-T318A on Hop1 phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-Hop1 antibody. Positions of the unphosphorylated and phosphorylated Hop1-species are indicated to the right of the blot. Shown on the right panels is quantification analysis of the Western images, where the signal in the ‘pHop1’ region in each lane is divided by the total signal (‘pHop1’+ ‘Hop1’) in the corresponding lane.
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pone.0134297.g003: Hop1-S298 phosphorylation is required for dmc1Δ-dependent meiotic checkpoint response.(A) and (B). Fraction of cells undergone meiosis I (MI) in synchronous meiotic cultures of HOP1, hop1-S298A, and hop1-T318A at 23°C in a DMC1 or dmc1Δ background. T50: Time at which 50% of the active culture has completed MI. At least three separate timecourses were considered, for each set of strains for each background. T50 kinetics were calculated from the most representative timecourse for each set of strain and DMC1/dmc1 background. Errors were calculated from the 95% confidence interval of a binomial distribution. (C) and (D). Effects of hop1-S298A and hop1-T318A on Mek1-HA phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-HA antibody. Positions of the unphosphorylated and phosphorylated Mek1-HA species are indicated to the right of the blot. ‘% pMek’ corresponds to the proportion of phosphorylated Mek1-HA species in each lane calculated by dividing the signal found in the ‘pMek1-HA’ area of the gel by the total signal (‘pMek1-HA’ + ‘Mek1-HA’). (E) and (F). Effects of hop1-S298A and hop1-T318A on Hop1 phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-Hop1 antibody. Positions of the unphosphorylated and phosphorylated Hop1-species are indicated to the right of the blot. Shown on the right panels is quantification analysis of the Western images, where the signal in the ‘pHop1’ region in each lane is divided by the total signal (‘pHop1’+ ‘Hop1’) in the corresponding lane.

Mentions: Genetic evidence above suggests that the Hop1 phospho-S298 plays an auxiliary role, along with the essential phosho-T318, to promote spore viability and mediate dmc1Δ meiotic arrest. We wished to address the molecular basis of its function. Since an essential function of the Tel1/Mec1 phosphorylation of Hop1 is to activate Mek1 [6], we proceeded to assess the effects of hop1-S298A on Mek1 phosphorylation. In a HOP1 strain during normal meiosis, Mek1 phosphorylation was modest and transient, observed at 4 and 6 hours (Fig 3C). Comparable levels of Mek1 phosphorylation, reaching ~24% of total Mek1-HA signal at t = 6 hours, were observed in hop1-S298A cells (Fig 3C). As shown previously [6, 20], no Mek1 activation was observed in a hop1-T318A background. We conclude that the Hop1 phospho-S298, unlike the phospho-T318, is dispensable for the essential Mek1 activation during unchallenged meiosis.


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)

Hop1-S298 phosphorylation is required for dmc1Δ-dependent meiotic checkpoint response.(A) and (B). Fraction of cells undergone meiosis I (MI) in synchronous meiotic cultures of HOP1, hop1-S298A, and hop1-T318A at 23°C in a DMC1 or dmc1Δ background. T50: Time at which 50% of the active culture has completed MI. At least three separate timecourses were considered, for each set of strains for each background. T50 kinetics were calculated from the most representative timecourse for each set of strain and DMC1/dmc1 background. Errors were calculated from the 95% confidence interval of a binomial distribution. (C) and (D). Effects of hop1-S298A and hop1-T318A on Mek1-HA phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-HA antibody. Positions of the unphosphorylated and phosphorylated Mek1-HA species are indicated to the right of the blot. ‘% pMek’ corresponds to the proportion of phosphorylated Mek1-HA species in each lane calculated by dividing the signal found in the ‘pMek1-HA’ area of the gel by the total signal (‘pMek1-HA’ + ‘Mek1-HA’). (E) and (F). Effects of hop1-S298A and hop1-T318A on Hop1 phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-Hop1 antibody. Positions of the unphosphorylated and phosphorylated Hop1-species are indicated to the right of the blot. Shown on the right panels is quantification analysis of the Western images, where the signal in the ‘pHop1’ region in each lane is divided by the total signal (‘pHop1’+ ‘Hop1’) in the corresponding lane.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520594&req=5

pone.0134297.g003: Hop1-S298 phosphorylation is required for dmc1Δ-dependent meiotic checkpoint response.(A) and (B). Fraction of cells undergone meiosis I (MI) in synchronous meiotic cultures of HOP1, hop1-S298A, and hop1-T318A at 23°C in a DMC1 or dmc1Δ background. T50: Time at which 50% of the active culture has completed MI. At least three separate timecourses were considered, for each set of strains for each background. T50 kinetics were calculated from the most representative timecourse for each set of strain and DMC1/dmc1 background. Errors were calculated from the 95% confidence interval of a binomial distribution. (C) and (D). Effects of hop1-S298A and hop1-T318A on Mek1-HA phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-HA antibody. Positions of the unphosphorylated and phosphorylated Mek1-HA species are indicated to the right of the blot. ‘% pMek’ corresponds to the proportion of phosphorylated Mek1-HA species in each lane calculated by dividing the signal found in the ‘pMek1-HA’ area of the gel by the total signal (‘pMek1-HA’ + ‘Mek1-HA’). (E) and (F). Effects of hop1-S298A and hop1-T318A on Hop1 phosphorylation during DMC1 and dmc1Δ meiosis. Samples from the cultures described in panels (A) and (B) were subjected to Western blot analysis using anti-Hop1 antibody. Positions of the unphosphorylated and phosphorylated Hop1-species are indicated to the right of the blot. Shown on the right panels is quantification analysis of the Western images, where the signal in the ‘pHop1’ region in each lane is divided by the total signal (‘pHop1’+ ‘Hop1’) in the corresponding lane.
Mentions: Genetic evidence above suggests that the Hop1 phospho-S298 plays an auxiliary role, along with the essential phosho-T318, to promote spore viability and mediate dmc1Δ meiotic arrest. We wished to address the molecular basis of its function. Since an essential function of the Tel1/Mec1 phosphorylation of Hop1 is to activate Mek1 [6], we proceeded to assess the effects of hop1-S298A on Mek1 phosphorylation. In a HOP1 strain during normal meiosis, Mek1 phosphorylation was modest and transient, observed at 4 and 6 hours (Fig 3C). Comparable levels of Mek1 phosphorylation, reaching ~24% of total Mek1-HA signal at t = 6 hours, were observed in hop1-S298A cells (Fig 3C). As shown previously [6, 20], no Mek1 activation was observed in a hop1-T318A background. We conclude that the Hop1 phospho-S298, unlike the phospho-T318, is dispensable for the essential Mek1 activation during unchallenged meiosis.

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