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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

Eso1 and Rev1 were associated in G1 phase.A,eso1Δpolh and rev1Δ mutants exhibited similar sensitivities to UV irradiation. Cells in the logarithmic growth phase were serially diluted by 5 fold. Cells were then spotted on YES plates and exposed to UV light (0, 100, or 150 J/m2). Plates were incubated at 30°C, and the growth of wt, eso1Δpolh, and rev1Δ strains was observed 3 days after the irradiation. B, Eso1 associated with Rev1 in cdc10-arrested extracts. cdc10 rev1flag eso1V5 and cdc25 rev1flag eso1V5 strains were first grown at 25°C, and the cultures were split into two. The first half was further grown at 25°C, and the second half was grown at 36°C for 3 h. The cells were harvested, and whole cell extracts were prepared. Immunoprecipitation was then carried out using anti-flag or anti-V5 antibodies. The panels represent input, Flag IP, and V5 IP of Rev1 and Eso1 protein in the cdc10 strain at 25°C, the cdc10 strain at 36.5°C, the cdc25 strain at 25°C, and the cdc25 strain at 36.5°C. C, Eso1 was coprecipitated with Rev7. eso1myc and eso1myc rev7flag strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions. D, Rev7 failed to coprecipitate Eso1 in the absence of Rev1. eso1myc rev7flag and eso1myc rev7flag rev1Δ strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions.
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pone.0130000.g005: Eso1 and Rev1 were associated in G1 phase.A,eso1Δpolh and rev1Δ mutants exhibited similar sensitivities to UV irradiation. Cells in the logarithmic growth phase were serially diluted by 5 fold. Cells were then spotted on YES plates and exposed to UV light (0, 100, or 150 J/m2). Plates were incubated at 30°C, and the growth of wt, eso1Δpolh, and rev1Δ strains was observed 3 days after the irradiation. B, Eso1 associated with Rev1 in cdc10-arrested extracts. cdc10 rev1flag eso1V5 and cdc25 rev1flag eso1V5 strains were first grown at 25°C, and the cultures were split into two. The first half was further grown at 25°C, and the second half was grown at 36°C for 3 h. The cells were harvested, and whole cell extracts were prepared. Immunoprecipitation was then carried out using anti-flag or anti-V5 antibodies. The panels represent input, Flag IP, and V5 IP of Rev1 and Eso1 protein in the cdc10 strain at 25°C, the cdc10 strain at 36.5°C, the cdc25 strain at 25°C, and the cdc25 strain at 36.5°C. C, Eso1 was coprecipitated with Rev7. eso1myc and eso1myc rev7flag strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions. D, Rev7 failed to coprecipitate Eso1 in the absence of Rev1. eso1myc rev7flag and eso1myc rev7flag rev1Δ strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions.

Mentions: Studies investigating the function of Rev1 in humans and budding yeast have indicated that Rev1 may serve as a scaffold for TLS polymerases [32, 52–54]. The increase in Rev1 protein levels during G1 phase may be a prerequisite for the formation of the TLS polymerase complex in S phase, similar to the role of Cdc18 in replicative complexes. To assess this possibility, we examined the relationship between Rev1 and Eso1, a polymerase η homolog. First, we examined the UV sensitivity of strains deficient in Rev1 or polymerase η. The rev1Δ mutant conferred cells with a sensitivity similar to that of eso1Δpolη when cells were irradiated at 100 or 150 J/m2 (Fig 5A). The enzymatic function of Rev1 after UV irradiation is not thought to be critical since Rev1 preferentially functions as a dCMP transferase. Therefore, this result suggested that Rev1 served as a regulator of Eso1. Next, we examined the physical interactions between Rev1 and Eso1. When we immunoprecipitated Rev1 or Eso1 from extracts of cdc10-arrested cells, Eso1 was co-immunoprecipitated with Rev1, and Rev1 was co-immunoprecipitated with Eso1 (Fig 5B). Therefore, these data confirmed the physical association between Rev1 and Eso1 in G1 phase. Next, we examined whether Rev1, polymerase η, and polymerase ζ formed a complex. As reported in other species [55], Rev1 was associated with the polζ subunit Rev7 (S2 Fig). Next, we examined the association of Eso1 with Rev7 in the rev1 deletion background. As shown in Fig 5C, immunoprecipitation analysis showed that Eso1 associated with Rev7. However, we were unable to detect this association by immunoprecipitation in the rev1Δ background (Fig 5D). From these results, we concluded that the fission yeast Rev1 also functioned as a polζ assembly factor for Eso1/polη.


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)

Eso1 and Rev1 were associated in G1 phase.A,eso1Δpolh and rev1Δ mutants exhibited similar sensitivities to UV irradiation. Cells in the logarithmic growth phase were serially diluted by 5 fold. Cells were then spotted on YES plates and exposed to UV light (0, 100, or 150 J/m2). Plates were incubated at 30°C, and the growth of wt, eso1Δpolh, and rev1Δ strains was observed 3 days after the irradiation. B, Eso1 associated with Rev1 in cdc10-arrested extracts. cdc10 rev1flag eso1V5 and cdc25 rev1flag eso1V5 strains were first grown at 25°C, and the cultures were split into two. The first half was further grown at 25°C, and the second half was grown at 36°C for 3 h. The cells were harvested, and whole cell extracts were prepared. Immunoprecipitation was then carried out using anti-flag or anti-V5 antibodies. The panels represent input, Flag IP, and V5 IP of Rev1 and Eso1 protein in the cdc10 strain at 25°C, the cdc10 strain at 36.5°C, the cdc25 strain at 25°C, and the cdc25 strain at 36.5°C. C, Eso1 was coprecipitated with Rev7. eso1myc and eso1myc rev7flag strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions. D, Rev7 failed to coprecipitate Eso1 in the absence of Rev1. eso1myc rev7flag and eso1myc rev7flag rev1Δ strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions.
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Related In: Results  -  Collection

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pone.0130000.g005: Eso1 and Rev1 were associated in G1 phase.A,eso1Δpolh and rev1Δ mutants exhibited similar sensitivities to UV irradiation. Cells in the logarithmic growth phase were serially diluted by 5 fold. Cells were then spotted on YES plates and exposed to UV light (0, 100, or 150 J/m2). Plates were incubated at 30°C, and the growth of wt, eso1Δpolh, and rev1Δ strains was observed 3 days after the irradiation. B, Eso1 associated with Rev1 in cdc10-arrested extracts. cdc10 rev1flag eso1V5 and cdc25 rev1flag eso1V5 strains were first grown at 25°C, and the cultures were split into two. The first half was further grown at 25°C, and the second half was grown at 36°C for 3 h. The cells were harvested, and whole cell extracts were prepared. Immunoprecipitation was then carried out using anti-flag or anti-V5 antibodies. The panels represent input, Flag IP, and V5 IP of Rev1 and Eso1 protein in the cdc10 strain at 25°C, the cdc10 strain at 36.5°C, the cdc25 strain at 25°C, and the cdc25 strain at 36.5°C. C, Eso1 was coprecipitated with Rev7. eso1myc and eso1myc rev7flag strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions. D, Rev7 failed to coprecipitate Eso1 in the absence of Rev1. eso1myc rev7flag and eso1myc rev7flag rev1Δ strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-flag antibodies. The panels show the expression of Eso1 and Rev7 in input samples and immunoprecipitated fractions.
Mentions: Studies investigating the function of Rev1 in humans and budding yeast have indicated that Rev1 may serve as a scaffold for TLS polymerases [32, 52–54]. The increase in Rev1 protein levels during G1 phase may be a prerequisite for the formation of the TLS polymerase complex in S phase, similar to the role of Cdc18 in replicative complexes. To assess this possibility, we examined the relationship between Rev1 and Eso1, a polymerase η homolog. First, we examined the UV sensitivity of strains deficient in Rev1 or polymerase η. The rev1Δ mutant conferred cells with a sensitivity similar to that of eso1Δpolη when cells were irradiated at 100 or 150 J/m2 (Fig 5A). The enzymatic function of Rev1 after UV irradiation is not thought to be critical since Rev1 preferentially functions as a dCMP transferase. Therefore, this result suggested that Rev1 served as a regulator of Eso1. Next, we examined the physical interactions between Rev1 and Eso1. When we immunoprecipitated Rev1 or Eso1 from extracts of cdc10-arrested cells, Eso1 was co-immunoprecipitated with Rev1, and Rev1 was co-immunoprecipitated with Eso1 (Fig 5B). Therefore, these data confirmed the physical association between Rev1 and Eso1 in G1 phase. Next, we examined whether Rev1, polymerase η, and polymerase ζ formed a complex. As reported in other species [55], Rev1 was associated with the polζ subunit Rev7 (S2 Fig). Next, we examined the association of Eso1 with Rev7 in the rev1 deletion background. As shown in Fig 5C, immunoprecipitation analysis showed that Eso1 associated with Rev7. However, we were unable to detect this association by immunoprecipitation in the rev1Δ background (Fig 5D). From these results, we concluded that the fission yeast Rev1 also functioned as a polζ assembly factor for Eso1/polη.

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