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The CDK-activating kinase (CAK) Csk1 is required for normal levels of homologous recombination and resistance to DNA damage in fission yeast.

Gerber HB, Pikman Y, Fisher RP - PLoS ONE (2008)

Bottom Line: Csk1 is required for normal levels of homologous recombination (HR), and interacts genetically with components of the HR pathway.The two CAKs in fission yeast, which differ with respect to their substrate range and preferences for monomeric CDKs versus CDK/cyclin complexes as substrates, also support different functions of the CDK network in vivo.Csk1 plays a non-redundant role in safeguarding genomic integrity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

ABSTRACT

Background: Cyclin-dependent kinases (CDKs) perform essential roles in cell division and gene expression in all eukaryotes. The requirement for an upstream CDK-activating kinase (CAK) is also universally conserved, but the fission yeast Schizosaccharomyces pombe appears to be unique in having two CAKs with both overlapping and specialized functions that can be dissected genetically. The Mcs6 complex--orthologous to metazoan Cdk7/cyclin H/Mat1--activates the cell-cycle CDK, Cdk1, but its non-redundant essential function appears to be in regulation of gene expression, as part of transcription factor TFIIH. The other CAK is Csk1, an ortholog of budding yeast Cak1, which activates all three essential CDKs in S. pombe--Cdk1, Mcs6 and Cdk9, the catalytic subunit of positive transcription elongation factor b (P-TEFb)--but is not itself essential.

Methodology/principal findings: Cells lacking csk1(+) are viable but hypersensitive to agents that damage DNA or block replication. Csk1 is required for normal levels of homologous recombination (HR), and interacts genetically with components of the HR pathway. Tests of damage sensitivity in csk1, mcs6 and cdk9 mutants indicate that Csk1 acts pleiotropically, through Cdk9 and at least one other target (but not through Mcs6) to preserve genomic integrity.

Conclusions/significance: The two CAKs in fission yeast, which differ with respect to their substrate range and preferences for monomeric CDKs versus CDK/cyclin complexes as substrates, also support different functions of the CDK network in vivo. Csk1 plays a non-redundant role in safeguarding genomic integrity. We propose that specialized activation pathways dependent on different CAKs might insulate CDK functions important in DNA damage responses from those capable of triggering mitosis.

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Related in: MedlinePlus

Multiple Csk1 targets contribute to the csk1Δ UV-sensitivity and growth phenotypes.Survival after UV irradiation of the following strains: (A) wild type (JS78), csk1Δ (JS155), mcs6-HA (JS167), mcs6S165A-HA (JS207); (B) wild type (JS78), csk1Δ (JS155), cdk9T212A (HD7-24), csk1Δ cdk9T212A (HD7-44); and (C) wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133). (D) Spot assays on YES and EMM plates with the following strains: wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133).
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pone-0001492-g005: Multiple Csk1 targets contribute to the csk1Δ UV-sensitivity and growth phenotypes.Survival after UV irradiation of the following strains: (A) wild type (JS78), csk1Δ (JS155), mcs6-HA (JS167), mcs6S165A-HA (JS207); (B) wild type (JS78), csk1Δ (JS155), cdk9T212A (HD7-24), csk1Δ cdk9T212A (HD7-44); and (C) wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133). (D) Spot assays on YES and EMM plates with the following strains: wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133).

Mentions: Csk1 promotes survival of DNA damage, presumably through one or more downstream CDKs. To ascertain which kinase(s) might require activation by Csk1 to perform a function in the DNA damage response, we mutated the sites phosphorylated by Csk1 in two essential S. pombe CDKs, Mcs6 and Cdk9, and tested the resulting mutants for DNA damage-sensitivity. The addition of a hemagglutinin (HA) epitope to the carboxyl terminus of Mcs6 caused a mild UV-hypersensitivity phenotype, which was not further exacerbated by mutation of the T-loop phosphorylation site, Ser165, to Ala; the mcs6S165A-HA strain had a UV sensitivity similar to that of tagged but otherwise wild-type mcs6-HA (Fig. 5A). This suggests the mcs6S165A mutation by itself did not increase UV-sensitivity, and that failure to activate Mcs6 fully is unlikely to contribute to the DNA damage-sensitivity of a csk1Δ strain.


The CDK-activating kinase (CAK) Csk1 is required for normal levels of homologous recombination and resistance to DNA damage in fission yeast.

Gerber HB, Pikman Y, Fisher RP - PLoS ONE (2008)

Multiple Csk1 targets contribute to the csk1Δ UV-sensitivity and growth phenotypes.Survival after UV irradiation of the following strains: (A) wild type (JS78), csk1Δ (JS155), mcs6-HA (JS167), mcs6S165A-HA (JS207); (B) wild type (JS78), csk1Δ (JS155), cdk9T212A (HD7-24), csk1Δ cdk9T212A (HD7-44); and (C) wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133). (D) Spot assays on YES and EMM plates with the following strains: wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2200797&req=5

pone-0001492-g005: Multiple Csk1 targets contribute to the csk1Δ UV-sensitivity and growth phenotypes.Survival after UV irradiation of the following strains: (A) wild type (JS78), csk1Δ (JS155), mcs6-HA (JS167), mcs6S165A-HA (JS207); (B) wild type (JS78), csk1Δ (JS155), cdk9T212A (HD7-24), csk1Δ cdk9T212A (HD7-44); and (C) wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133). (D) Spot assays on YES and EMM plates with the following strains: wild type (JS78), csk1Δ (JS155), cdk9T212E (HG127), csk1Δ cdk9T212E (HG133).
Mentions: Csk1 promotes survival of DNA damage, presumably through one or more downstream CDKs. To ascertain which kinase(s) might require activation by Csk1 to perform a function in the DNA damage response, we mutated the sites phosphorylated by Csk1 in two essential S. pombe CDKs, Mcs6 and Cdk9, and tested the resulting mutants for DNA damage-sensitivity. The addition of a hemagglutinin (HA) epitope to the carboxyl terminus of Mcs6 caused a mild UV-hypersensitivity phenotype, which was not further exacerbated by mutation of the T-loop phosphorylation site, Ser165, to Ala; the mcs6S165A-HA strain had a UV sensitivity similar to that of tagged but otherwise wild-type mcs6-HA (Fig. 5A). This suggests the mcs6S165A mutation by itself did not increase UV-sensitivity, and that failure to activate Mcs6 fully is unlikely to contribute to the DNA damage-sensitivity of a csk1Δ strain.

Bottom Line: Csk1 is required for normal levels of homologous recombination (HR), and interacts genetically with components of the HR pathway.The two CAKs in fission yeast, which differ with respect to their substrate range and preferences for monomeric CDKs versus CDK/cyclin complexes as substrates, also support different functions of the CDK network in vivo.Csk1 plays a non-redundant role in safeguarding genomic integrity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

ABSTRACT

Background: Cyclin-dependent kinases (CDKs) perform essential roles in cell division and gene expression in all eukaryotes. The requirement for an upstream CDK-activating kinase (CAK) is also universally conserved, but the fission yeast Schizosaccharomyces pombe appears to be unique in having two CAKs with both overlapping and specialized functions that can be dissected genetically. The Mcs6 complex--orthologous to metazoan Cdk7/cyclin H/Mat1--activates the cell-cycle CDK, Cdk1, but its non-redundant essential function appears to be in regulation of gene expression, as part of transcription factor TFIIH. The other CAK is Csk1, an ortholog of budding yeast Cak1, which activates all three essential CDKs in S. pombe--Cdk1, Mcs6 and Cdk9, the catalytic subunit of positive transcription elongation factor b (P-TEFb)--but is not itself essential.

Methodology/principal findings: Cells lacking csk1(+) are viable but hypersensitive to agents that damage DNA or block replication. Csk1 is required for normal levels of homologous recombination (HR), and interacts genetically with components of the HR pathway. Tests of damage sensitivity in csk1, mcs6 and cdk9 mutants indicate that Csk1 acts pleiotropically, through Cdk9 and at least one other target (but not through Mcs6) to preserve genomic integrity.

Conclusions/significance: The two CAKs in fission yeast, which differ with respect to their substrate range and preferences for monomeric CDKs versus CDK/cyclin complexes as substrates, also support different functions of the CDK network in vivo. Csk1 plays a non-redundant role in safeguarding genomic integrity. We propose that specialized activation pathways dependent on different CAKs might insulate CDK functions important in DNA damage responses from those capable of triggering mitosis.

Show MeSH
Related in: MedlinePlus