Limits...
The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress.

Soriano-Carot M, Bañó MC, Igual JC - Cell Div (2012)

Bottom Line: However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress.The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, 46100 Burjassot (Valencia), Spain. jcigual@uv.es.

ABSTRACT

Background: The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism.

Results: This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.

Conclusions: Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

No MeSH data available.


Related in: MedlinePlus

Hypersensitivity of slt2 mutant strain to genotoxic stress. A) 10-fold serial dilutions from exponentially growing cultures of wild type (W303-1a) and slt2 (JCY1062) strains were spotted onto YPD medium containing 100 mM hydroxyurea, 0.025% MMS or 5 μg/mL phleomycin or were exposed to UV radiation (35 J/m2). Plates were incubated at 25°C for 3 days. B) Aliquots from exponentially growing cultures of wild type (W303-1a), slt2 (JCY1062) and mec1 sml1 (JCY1039) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined. C) Aliquots from exponentially growing cultures of wild type (SEY6211) and slt2 (JCY193) strains were incubated in the presence of 0.4 M HU, 0.02% MMS, 5 μg/mL phleomycin for 1 hour or were exposed to UV radiation (10 J/m2). Cell survival relative to untreated controls was determined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3298782&req=5

Figure 1: Hypersensitivity of slt2 mutant strain to genotoxic stress. A) 10-fold serial dilutions from exponentially growing cultures of wild type (W303-1a) and slt2 (JCY1062) strains were spotted onto YPD medium containing 100 mM hydroxyurea, 0.025% MMS or 5 μg/mL phleomycin or were exposed to UV radiation (35 J/m2). Plates were incubated at 25°C for 3 days. B) Aliquots from exponentially growing cultures of wild type (W303-1a), slt2 (JCY1062) and mec1 sml1 (JCY1039) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined. C) Aliquots from exponentially growing cultures of wild type (SEY6211) and slt2 (JCY193) strains were incubated in the presence of 0.4 M HU, 0.02% MMS, 5 μg/mL phleomycin for 1 hour or were exposed to UV radiation (10 J/m2). Cell survival relative to untreated controls was determined.

Mentions: Cells must cope with different genotoxic stresses to guarantee genomic integrity. The nature and form of action of these genotoxic stresses notably differ. Treatment with hydroxyurea (HU) inhibits ribonucleotide reductase, causing a depletion of dNTP pools, which interferes with DNA replication fork progression and originates subsequent chromosome breakages. Previous work from our group demonstrated that slt2 mutant strain growth in the presence of HU is severely affected [43]. We wondered whether Slt2 could also be related to other types of DNA damage apart from replication blockage. To investigate this possibility, slt2 mutant strain growth was assayed under conditions that induce the methylation of bases (incubation with alkylating agent methylmetanosulfonate -MMS-), the covalent cross-linking of adjacent pyrimidine bases (irradiation with UV light) or double-strand breaks (incubation with phleomycin). As Figure 1A illustrates, the slt2 mutant strain was unable to properly grow when compared to the wild-type strain, not only in the presence of HU, but also in the presence of MMS, phleomycin, or even after UV irradiation. Quantitative survival assays with various doses of genotoxic treatments confirmed an increased loss of cell viability in the absence of Slt2 (Figure 1B). The sensitivity of slt2 cells to genotoxic stress was less severe than the one observed in the DNA-damage checkpoint mutant mec1 (Figure 1B). The original W303-1a strain contains the rad5-535 mutation, which could contribute to the observed growth defects. Therefore, growth analysis was also carried out in a RAD5 independent genetic background. As it is shown in Figure 1C, Slt2 inactivation in the SEY6211 strain also originated a reduced cell viability. All these results indicate that yeast cells need a functional Slt2 MAP kinase to optimally survive DNA damage, whatever the nature of the damage, suggesting that Slt2 plays a central role in the cellular response to genotoxic stress.


The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress.

Soriano-Carot M, Bañó MC, Igual JC - Cell Div (2012)

Hypersensitivity of slt2 mutant strain to genotoxic stress. A) 10-fold serial dilutions from exponentially growing cultures of wild type (W303-1a) and slt2 (JCY1062) strains were spotted onto YPD medium containing 100 mM hydroxyurea, 0.025% MMS or 5 μg/mL phleomycin or were exposed to UV radiation (35 J/m2). Plates were incubated at 25°C for 3 days. B) Aliquots from exponentially growing cultures of wild type (W303-1a), slt2 (JCY1062) and mec1 sml1 (JCY1039) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined. C) Aliquots from exponentially growing cultures of wild type (SEY6211) and slt2 (JCY193) strains were incubated in the presence of 0.4 M HU, 0.02% MMS, 5 μg/mL phleomycin for 1 hour or were exposed to UV radiation (10 J/m2). Cell survival relative to untreated controls was determined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298782&req=5

Figure 1: Hypersensitivity of slt2 mutant strain to genotoxic stress. A) 10-fold serial dilutions from exponentially growing cultures of wild type (W303-1a) and slt2 (JCY1062) strains were spotted onto YPD medium containing 100 mM hydroxyurea, 0.025% MMS or 5 μg/mL phleomycin or were exposed to UV radiation (35 J/m2). Plates were incubated at 25°C for 3 days. B) Aliquots from exponentially growing cultures of wild type (W303-1a), slt2 (JCY1062) and mec1 sml1 (JCY1039) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined. C) Aliquots from exponentially growing cultures of wild type (SEY6211) and slt2 (JCY193) strains were incubated in the presence of 0.4 M HU, 0.02% MMS, 5 μg/mL phleomycin for 1 hour or were exposed to UV radiation (10 J/m2). Cell survival relative to untreated controls was determined.
Mentions: Cells must cope with different genotoxic stresses to guarantee genomic integrity. The nature and form of action of these genotoxic stresses notably differ. Treatment with hydroxyurea (HU) inhibits ribonucleotide reductase, causing a depletion of dNTP pools, which interferes with DNA replication fork progression and originates subsequent chromosome breakages. Previous work from our group demonstrated that slt2 mutant strain growth in the presence of HU is severely affected [43]. We wondered whether Slt2 could also be related to other types of DNA damage apart from replication blockage. To investigate this possibility, slt2 mutant strain growth was assayed under conditions that induce the methylation of bases (incubation with alkylating agent methylmetanosulfonate -MMS-), the covalent cross-linking of adjacent pyrimidine bases (irradiation with UV light) or double-strand breaks (incubation with phleomycin). As Figure 1A illustrates, the slt2 mutant strain was unable to properly grow when compared to the wild-type strain, not only in the presence of HU, but also in the presence of MMS, phleomycin, or even after UV irradiation. Quantitative survival assays with various doses of genotoxic treatments confirmed an increased loss of cell viability in the absence of Slt2 (Figure 1B). The sensitivity of slt2 cells to genotoxic stress was less severe than the one observed in the DNA-damage checkpoint mutant mec1 (Figure 1B). The original W303-1a strain contains the rad5-535 mutation, which could contribute to the observed growth defects. Therefore, growth analysis was also carried out in a RAD5 independent genetic background. As it is shown in Figure 1C, Slt2 inactivation in the SEY6211 strain also originated a reduced cell viability. All these results indicate that yeast cells need a functional Slt2 MAP kinase to optimally survive DNA damage, whatever the nature of the damage, suggesting that Slt2 plays a central role in the cellular response to genotoxic stress.

Bottom Line: However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress.The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, 46100 Burjassot (Valencia), Spain. jcigual@uv.es.

ABSTRACT

Background: The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism.

Results: This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.

Conclusions: Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

No MeSH data available.


Related in: MedlinePlus