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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

Analysis of ribonucleotide reductase (RNR) function in the slt2 mutant strain. A) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. The protein level of the different subunits of the RNR enzyme was analyzed by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. B) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 0,04% MMS. The cellular content of dATP, dCTP and dGTP were determined in crude cell extracts. Similar results were obtained with cells incubated for 60 min in the presence of HU or MMS.
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Figure 6: Analysis of ribonucleotide reductase (RNR) function in the slt2 mutant strain. A) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. The protein level of the different subunits of the RNR enzyme was analyzed by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. B) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 0,04% MMS. The cellular content of dATP, dCTP and dGTP were determined in crude cell extracts. Similar results were obtained with cells incubated for 60 min in the presence of HU or MMS.

Mentions: In an initial approach to characterize whether Slt2 could affect dNTP pools, we first analyzed the ribonucleotide reductase protein levels in slt2 cells in normal conditions or after induction of DNA damage. A Western blot analysis revealed that all the ribonucleotide reductase subunits were expressed at a similar level in wild-type and slt2 cells both before and after HU or MMS treatments (Figure 6A). It is possible that ribonucleotide reductase activity could be defective in slt2 mutant cells despite the amount of ribonucleotide reductase enzyme not being altered. To test this, we measured the cellular content of dATP, dCTP and dGTP in the wild-type and slt2 mutant strains. As observed in Figure 6B, inactivation of Slt2 caused no significant changes in the concentration of the three dNTPs under both basal conditions and in MMS-treated cells. This result demonstrates that Slt2 is not involved in the control of dNTP pools.


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)

Analysis of ribonucleotide reductase (RNR) function in the slt2 mutant strain. A) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. The protein level of the different subunits of the RNR enzyme was analyzed by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. B) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 0,04% MMS. The cellular content of dATP, dCTP and dGTP were determined in crude cell extracts. Similar results were obtained with cells incubated for 60 min in the presence of HU or MMS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Analysis of ribonucleotide reductase (RNR) function in the slt2 mutant strain. A) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. The protein level of the different subunits of the RNR enzyme was analyzed by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. B) Exponentially growing cultures of the wild type W303-1a strain were split and incubated for 120 min in the absence or presence of 0,04% MMS. The cellular content of dATP, dCTP and dGTP were determined in crude cell extracts. Similar results were obtained with cells incubated for 60 min in the presence of HU or MMS.
Mentions: In an initial approach to characterize whether Slt2 could affect dNTP pools, we first analyzed the ribonucleotide reductase protein levels in slt2 cells in normal conditions or after induction of DNA damage. A Western blot analysis revealed that all the ribonucleotide reductase subunits were expressed at a similar level in wild-type and slt2 cells both before and after HU or MMS treatments (Figure 6A). It is possible that ribonucleotide reductase activity could be defective in slt2 mutant cells despite the amount of ribonucleotide reductase enzyme not being altered. To test this, we measured the cellular content of dATP, dCTP and dGTP in the wild-type and slt2 mutant strains. As observed in Figure 6B, inactivation of Slt2 caused no significant changes in the concentration of the three dNTPs under both basal conditions and in MMS-treated cells. This result demonstrates that Slt2 is not involved in the control of dNTP pools.

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