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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

Rev1 protein was most abundant in G1 phase.A, The TLS polymerases Eso1 and Kpa1 were upregulated in S phase. Eso1, Kpa1, and Rev7 protein expression levels were examined after cdc25-dependent block and release. Whole cell extracts were prepared by the boiling method, and western blotting was performed at different times (20–200 min) after the release. The graph represents the septation index after release. The lower panels show the expression patterns of Eso1, Kpa1, Rev7, Pcn1, Cdc13 (cyclin B), and Cdc2. B, Rev3 protein expression peaked during S phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The middle panel shows the expression pattern of Rev3, which was detected by anti-flag antibodies. The lower panel shows the protein expression of Pcn1 in the chromatin fraction. C, The protein amount of Rev1 was highest in G1 phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The panels show the expression patterns of Rev1, Cdc13, and Cdc2. D, The protein expression of Rev1 in cell cycle mutants. cdc10, cdc20, cdc22, cdc21, cdc17, and cdc25 mutants harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the boiling method. The amount of Rev1 was analyzed. E, Modified forms of the Rev1 protein were observed only in cdc20 or cdc22. cdc10, cdc20, cdc22, and cdc25 cells harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the LiNi method and then subjected to immunoprecipitation. Rev1 protein expression in each sample was roughly adjusted, and western blotting was performed.
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pone.0130000.g001: Rev1 protein was most abundant in G1 phase.A, The TLS polymerases Eso1 and Kpa1 were upregulated in S phase. Eso1, Kpa1, and Rev7 protein expression levels were examined after cdc25-dependent block and release. Whole cell extracts were prepared by the boiling method, and western blotting was performed at different times (20–200 min) after the release. The graph represents the septation index after release. The lower panels show the expression patterns of Eso1, Kpa1, Rev7, Pcn1, Cdc13 (cyclin B), and Cdc2. B, Rev3 protein expression peaked during S phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The middle panel shows the expression pattern of Rev3, which was detected by anti-flag antibodies. The lower panel shows the protein expression of Pcn1 in the chromatin fraction. C, The protein amount of Rev1 was highest in G1 phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The panels show the expression patterns of Rev1, Cdc13, and Cdc2. D, The protein expression of Rev1 in cell cycle mutants. cdc10, cdc20, cdc22, cdc21, cdc17, and cdc25 mutants harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the boiling method. The amount of Rev1 was analyzed. E, Modified forms of the Rev1 protein were observed only in cdc20 or cdc22. cdc10, cdc20, cdc22, and cdc25 cells harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the LiNi method and then subjected to immunoprecipitation. Rev1 protein expression in each sample was roughly adjusted, and western blotting was performed.

Mentions: To determine the role of TLS polymerases in regulating the cell cycle, we first analyzed the protein levels of TLS polymerases in cdc25-synchronized cultures. As shown in Fig 1A, the septation index reached a maximum at 120 min after the temperature shift. The peak of Eso1 (DNA polymerase η) expression was observed at 120 min, suggesting that Eso1 protein was most abundant during S phase. The peak of Kpa1 (DNA polymerase κ) expression was observed at 140–160 min, indicating that Kpa1 was abundant during S/G2 phase. Rev7, an accessory subunit of DNA polymerase ζ, remained constant throughout the cell cycle. Unlike Rev7, Rev3, the catalytic subunit of DNA polymerase ζ, was expressed at its highest level during S phase (Fig 1B).


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)

Rev1 protein was most abundant in G1 phase.A, The TLS polymerases Eso1 and Kpa1 were upregulated in S phase. Eso1, Kpa1, and Rev7 protein expression levels were examined after cdc25-dependent block and release. Whole cell extracts were prepared by the boiling method, and western blotting was performed at different times (20–200 min) after the release. The graph represents the septation index after release. The lower panels show the expression patterns of Eso1, Kpa1, Rev7, Pcn1, Cdc13 (cyclin B), and Cdc2. B, Rev3 protein expression peaked during S phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The middle panel shows the expression pattern of Rev3, which was detected by anti-flag antibodies. The lower panel shows the protein expression of Pcn1 in the chromatin fraction. C, The protein amount of Rev1 was highest in G1 phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The panels show the expression patterns of Rev1, Cdc13, and Cdc2. D, The protein expression of Rev1 in cell cycle mutants. cdc10, cdc20, cdc22, cdc21, cdc17, and cdc25 mutants harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the boiling method. The amount of Rev1 was analyzed. E, Modified forms of the Rev1 protein were observed only in cdc20 or cdc22. cdc10, cdc20, cdc22, and cdc25 cells harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the LiNi method and then subjected to immunoprecipitation. Rev1 protein expression in each sample was roughly adjusted, and western blotting was performed.
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pone.0130000.g001: Rev1 protein was most abundant in G1 phase.A, The TLS polymerases Eso1 and Kpa1 were upregulated in S phase. Eso1, Kpa1, and Rev7 protein expression levels were examined after cdc25-dependent block and release. Whole cell extracts were prepared by the boiling method, and western blotting was performed at different times (20–200 min) after the release. The graph represents the septation index after release. The lower panels show the expression patterns of Eso1, Kpa1, Rev7, Pcn1, Cdc13 (cyclin B), and Cdc2. B, Rev3 protein expression peaked during S phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The middle panel shows the expression pattern of Rev3, which was detected by anti-flag antibodies. The lower panel shows the protein expression of Pcn1 in the chromatin fraction. C, The protein amount of Rev1 was highest in G1 phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The panels show the expression patterns of Rev1, Cdc13, and Cdc2. D, The protein expression of Rev1 in cell cycle mutants. cdc10, cdc20, cdc22, cdc21, cdc17, and cdc25 mutants harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the boiling method. The amount of Rev1 was analyzed. E, Modified forms of the Rev1 protein were observed only in cdc20 or cdc22. cdc10, cdc20, cdc22, and cdc25 cells harboring flag-tagged rev1 were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the LiNi method and then subjected to immunoprecipitation. Rev1 protein expression in each sample was roughly adjusted, and western blotting was performed.
Mentions: To determine the role of TLS polymerases in regulating the cell cycle, we first analyzed the protein levels of TLS polymerases in cdc25-synchronized cultures. As shown in Fig 1A, the septation index reached a maximum at 120 min after the temperature shift. The peak of Eso1 (DNA polymerase η) expression was observed at 120 min, suggesting that Eso1 protein was most abundant during S phase. The peak of Kpa1 (DNA polymerase κ) expression was observed at 140–160 min, indicating that Kpa1 was abundant during S/G2 phase. Rev7, an accessory subunit of DNA polymerase ζ, remained constant throughout the cell cycle. Unlike Rev7, Rev3, the catalytic subunit of DNA polymerase ζ, was expressed at its highest level during S phase (Fig 1B).

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