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The Protein Level of Rev1, a TLS Polymerase in Fission Yeast, Is Strictly Regulated during the Cell Cycle and after DNA Damage.

Uchiyama M, Terunuma J, Hanaoka F - PLoS ONE (2015)

Bottom Line: Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome.Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage.This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Translesion DNA synthesis provides an alternative DNA replication mechanism when template DNA is damaged. In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and Polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis polymerases. The enzymatic characteristics and protein-protein interactions of these polymerases have been intensively characterized; however, how these proteins are regulated during the cell cycle remains unclear. Therefore, we examined the cell cycle oscillation of translesion polymerases. Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome. Temperature-sensitive proteasome mutants, such as mts2-U31 and mts3-U32, stabilized Rev1 protein when the temperature was shifted to the restrictive condition. In addition, deletion of pop1 or pop2, subunits of SCF ubiquitin ligase complexes, upregulated Rev1 protein levels. Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage. This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter. From these results, we hypothesize that translesion DNA synthesis is strictly controlled through Rev1 protein levels in order to avoid unwanted mutagenesis.

No MeSH data available.


Related in: MedlinePlus

The protein expression of Rev1 was upregulated in response to DNA damage.A, Rev1 protein expression after mutagen treatment. At the logarithmic growth phase, wt cells harboring flag-tagged rev1 were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested. Whole cell extracts were prepared by the boiling method, and protein levels were examined by western blotting. The upper panel represents flag-tagged Rev1, and the lower panel shows CBB staining of the membrane. B, The upregulation of Rev1 was dependent on Rad3. At the logarithmic growth phase, rev1flag and rev1flag rad3Δ strains were treated with 0, 1, 5, 10, or 25 μM cisplatin for 3 h. Cells were then harvested, and whole cell extracts were prepared by the boiling method. Extracts were then subjected to western blotting. The upper panels show Rev1 and CBB staining of the rev1flag strain, and the lower panels show those of the rev1flag rad3Δ strain. C, The promoter region was important for the upregulation of Rev1 after DNA damage. At the logarithmic growth phase, cam1-rev1 cells were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested, and whole cell extracts were prepared by the boiling method. Protein expression was then examined by western blotting. The upper panel shows Rev1, and the lower panel shows CBB staining.
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pone.0130000.g007: The protein expression of Rev1 was upregulated in response to DNA damage.A, Rev1 protein expression after mutagen treatment. At the logarithmic growth phase, wt cells harboring flag-tagged rev1 were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested. Whole cell extracts were prepared by the boiling method, and protein levels were examined by western blotting. The upper panel represents flag-tagged Rev1, and the lower panel shows CBB staining of the membrane. B, The upregulation of Rev1 was dependent on Rad3. At the logarithmic growth phase, rev1flag and rev1flag rad3Δ strains were treated with 0, 1, 5, 10, or 25 μM cisplatin for 3 h. Cells were then harvested, and whole cell extracts were prepared by the boiling method. Extracts were then subjected to western blotting. The upper panels show Rev1 and CBB staining of the rev1flag strain, and the lower panels show those of the rev1flag rad3Δ strain. C, The promoter region was important for the upregulation of Rev1 after DNA damage. At the logarithmic growth phase, cam1-rev1 cells were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested, and whole cell extracts were prepared by the boiling method. Protein expression was then examined by western blotting. The upper panel shows Rev1, and the lower panel shows CBB staining.

Mentions: Because we confirmed the proteasomal regulation of Rev1 and its significance, we next focused on the unexpected upregulation of Rev1 in cdc21- and cdc17-arrested cultures. Both genes are important for DNA replication. It is possible that DNA damage structures are created because of defects in these gene products. Therefore, we examined whether Rev1 protein expression was altered following induction of DNA damage. As we described above, Rev1 exhibited sensitivity to cisplatin (CDDP), MMS, and 4NQO (S7 Fig). However, Rev1 did not exhibit sensitivity to camptothecin (CPT; S7 Fig). Therefore, we used these DNA-damaging agents and hydroxyurea, which blocks DNA replication, to examine changes in Rev1 expression. As shown in Fig 7A, Rev1 protein was upregulated in all samples treated with DNA-damaging agents, but was not detected in the hydroxyurea-treated sample.


The Protein Level of Rev1, a TLS Polymerase in Fission Yeast, Is Strictly Regulated during the Cell Cycle and after DNA Damage.

Uchiyama M, Terunuma J, Hanaoka F - PLoS ONE (2015)

The protein expression of Rev1 was upregulated in response to DNA damage.A, Rev1 protein expression after mutagen treatment. At the logarithmic growth phase, wt cells harboring flag-tagged rev1 were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested. Whole cell extracts were prepared by the boiling method, and protein levels were examined by western blotting. The upper panel represents flag-tagged Rev1, and the lower panel shows CBB staining of the membrane. B, The upregulation of Rev1 was dependent on Rad3. At the logarithmic growth phase, rev1flag and rev1flag rad3Δ strains were treated with 0, 1, 5, 10, or 25 μM cisplatin for 3 h. Cells were then harvested, and whole cell extracts were prepared by the boiling method. Extracts were then subjected to western blotting. The upper panels show Rev1 and CBB staining of the rev1flag strain, and the lower panels show those of the rev1flag rad3Δ strain. C, The promoter region was important for the upregulation of Rev1 after DNA damage. At the logarithmic growth phase, cam1-rev1 cells were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested, and whole cell extracts were prepared by the boiling method. Protein expression was then examined by western blotting. The upper panel shows Rev1, and the lower panel shows CBB staining.
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Related In: Results  -  Collection

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pone.0130000.g007: The protein expression of Rev1 was upregulated in response to DNA damage.A, Rev1 protein expression after mutagen treatment. At the logarithmic growth phase, wt cells harboring flag-tagged rev1 were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested. Whole cell extracts were prepared by the boiling method, and protein levels were examined by western blotting. The upper panel represents flag-tagged Rev1, and the lower panel shows CBB staining of the membrane. B, The upregulation of Rev1 was dependent on Rad3. At the logarithmic growth phase, rev1flag and rev1flag rad3Δ strains were treated with 0, 1, 5, 10, or 25 μM cisplatin for 3 h. Cells were then harvested, and whole cell extracts were prepared by the boiling method. Extracts were then subjected to western blotting. The upper panels show Rev1 and CBB staining of the rev1flag strain, and the lower panels show those of the rev1flag rad3Δ strain. C, The promoter region was important for the upregulation of Rev1 after DNA damage. At the logarithmic growth phase, cam1-rev1 cells were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested, and whole cell extracts were prepared by the boiling method. Protein expression was then examined by western blotting. The upper panel shows Rev1, and the lower panel shows CBB staining.
Mentions: Because we confirmed the proteasomal regulation of Rev1 and its significance, we next focused on the unexpected upregulation of Rev1 in cdc21- and cdc17-arrested cultures. Both genes are important for DNA replication. It is possible that DNA damage structures are created because of defects in these gene products. Therefore, we examined whether Rev1 protein expression was altered following induction of DNA damage. As we described above, Rev1 exhibited sensitivity to cisplatin (CDDP), MMS, and 4NQO (S7 Fig). However, Rev1 did not exhibit sensitivity to camptothecin (CPT; S7 Fig). Therefore, we used these DNA-damaging agents and hydroxyurea, which blocks DNA replication, to examine changes in Rev1 expression. As shown in Fig 7A, Rev1 protein was upregulated in all samples treated with DNA-damaging agents, but was not detected in the hydroxyurea-treated sample.

Bottom Line: Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome.Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage.This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Translesion DNA synthesis provides an alternative DNA replication mechanism when template DNA is damaged. In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and Polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis polymerases. The enzymatic characteristics and protein-protein interactions of these polymerases have been intensively characterized; however, how these proteins are regulated during the cell cycle remains unclear. Therefore, we examined the cell cycle oscillation of translesion polymerases. Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome. Temperature-sensitive proteasome mutants, such as mts2-U31 and mts3-U32, stabilized Rev1 protein when the temperature was shifted to the restrictive condition. In addition, deletion of pop1 or pop2, subunits of SCF ubiquitin ligase complexes, upregulated Rev1 protein levels. Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage. This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter. From these results, we hypothesize that translesion DNA synthesis is strictly controlled through Rev1 protein levels in order to avoid unwanted mutagenesis.

No MeSH data available.


Related in: MedlinePlus