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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

Pop1 and Pop2 were responsible for the stability of Rev1.A,pop1 and pop2 deletion mutants stabilized Rev1 protein. wt, pop1Δ, and pop2Δ strains harboring flag-tagged rev1 and an untagged rev1 wt strain were grown, and whole cell extracts were prepared by the boiling method. Protein expression levels were compared by western blotting. The upper panel shows the amount of Rev1 in rev1flag, rev1flag pop2Δ, rev1flag pop1Δ and untagged rev1 strains. The lower panel shows Cdc2 as a loading control. B, Rev1 was coprecipitated with Pop1. rev1flag pop1V5 and rev1flag strains were grown, and whole cell extracts were prepared by the LiNi method. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. C, Rev1 was co-precipitated with Pop2. The rev1flag pop2V5 strain was grown, and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop2 input. The right panels show Rev1 and Pop2 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions. D, The Rev1dK mutant was not coprecipitated with Pop1. rev1flag pop1V5 and rev1dKflag pop1V5 strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panel shows Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. E, Rev1KK (Rev1 761–818) associated with Pop1. Amino acids 761–818 of Rev1, which is the region deleted in Rev1dKK, were cloned into a pCAM1-flag expression vector. The pop1V5 strain with the pCAM1-rev1KKflag expression vector was grown at 30°C and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1KK and Pop1 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions.
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pone.0130000.g004: Pop1 and Pop2 were responsible for the stability of Rev1.A,pop1 and pop2 deletion mutants stabilized Rev1 protein. wt, pop1Δ, and pop2Δ strains harboring flag-tagged rev1 and an untagged rev1 wt strain were grown, and whole cell extracts were prepared by the boiling method. Protein expression levels were compared by western blotting. The upper panel shows the amount of Rev1 in rev1flag, rev1flag pop2Δ, rev1flag pop1Δ and untagged rev1 strains. The lower panel shows Cdc2 as a loading control. B, Rev1 was coprecipitated with Pop1. rev1flag pop1V5 and rev1flag strains were grown, and whole cell extracts were prepared by the LiNi method. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. C, Rev1 was co-precipitated with Pop2. The rev1flag pop2V5 strain was grown, and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop2 input. The right panels show Rev1 and Pop2 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions. D, The Rev1dK mutant was not coprecipitated with Pop1. rev1flag pop1V5 and rev1dKflag pop1V5 strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panel shows Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. E, Rev1KK (Rev1 761–818) associated with Pop1. Amino acids 761–818 of Rev1, which is the region deleted in Rev1dKK, were cloned into a pCAM1-flag expression vector. The pop1V5 strain with the pCAM1-rev1KKflag expression vector was grown at 30°C and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1KK and Pop1 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions.

Mentions: Next, we examined which ubiquitin ligase may be responsible for the degradation of Rev1. As shown in Fig 1C and 1D, Rev1 protein levels decreased dramatically at the onset of DNA replication. This pattern was similar to that of Cdc18, a functional homolog of budding yeast Cdc6 [49, 50]. The degradation of Cdc18 at G1/S depends on Pop1 and Pop2, subunits of SCF ubiquitin ligase [50, 51]. Therefore, we examined the involvement of Pop1 and Pop2 in the degradation of Rev1 at G1/S. Since pop1 and pop2 genes are not essential, we analyzed the protein levels of Rev1 in the pop1 or pop2 deletion background. The protein amount of Rev1 increased both in pop1Δ and pop2Δ mutants, although the contribution of pop1Δ was predominant (Fig 4A). Next, we examined the physical interactions between Rev1 and Pop1 or Pop2. As shown in Fig 4B and 4C, Rev1 was coprecipitated with Pop1 and Pop2. The interaction between Rev1 and Pop1 was also examined in cdc25-synchronized culture (S3 Fig). Rev1 was coprecipitated with Pop1, and the interaction peaked at about 100 min after the release. These data supported our hypothesis that Rev1 destruction at G1/S was controlled by SCF-dependent ubiquitination. To examine the contribution of the Lys-rich region of Rev1 for SCF-dependent proteolysis, we examined the protein-protein interactions between Pop1 and Rev1dK (Fig 4D). Interestingly, Rev1dK failed to interact with Pop1 following immunoprecipitation of Pop1. This result suggested that Rev1dK might become stable because it cannot interact with ubiquitin ligase. To test this hypothesis, we examined the interactions between Pop1 and Rev1KK (amino acids 761–818 of Rev1) (Fig 4E). Rev1KK was associated with Pop1. Based on these findings, we concluded that Rev1 destruction by the proteasome was mediated by Pop1 or Pop2 SCF ubiquitin ligase.


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)

Pop1 and Pop2 were responsible for the stability of Rev1.A,pop1 and pop2 deletion mutants stabilized Rev1 protein. wt, pop1Δ, and pop2Δ strains harboring flag-tagged rev1 and an untagged rev1 wt strain were grown, and whole cell extracts were prepared by the boiling method. Protein expression levels were compared by western blotting. The upper panel shows the amount of Rev1 in rev1flag, rev1flag pop2Δ, rev1flag pop1Δ and untagged rev1 strains. The lower panel shows Cdc2 as a loading control. B, Rev1 was coprecipitated with Pop1. rev1flag pop1V5 and rev1flag strains were grown, and whole cell extracts were prepared by the LiNi method. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. C, Rev1 was co-precipitated with Pop2. The rev1flag pop2V5 strain was grown, and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop2 input. The right panels show Rev1 and Pop2 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions. D, The Rev1dK mutant was not coprecipitated with Pop1. rev1flag pop1V5 and rev1dKflag pop1V5 strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panel shows Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. E, Rev1KK (Rev1 761–818) associated with Pop1. Amino acids 761–818 of Rev1, which is the region deleted in Rev1dKK, were cloned into a pCAM1-flag expression vector. The pop1V5 strain with the pCAM1-rev1KKflag expression vector was grown at 30°C and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1KK and Pop1 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions.
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pone.0130000.g004: Pop1 and Pop2 were responsible for the stability of Rev1.A,pop1 and pop2 deletion mutants stabilized Rev1 protein. wt, pop1Δ, and pop2Δ strains harboring flag-tagged rev1 and an untagged rev1 wt strain were grown, and whole cell extracts were prepared by the boiling method. Protein expression levels were compared by western blotting. The upper panel shows the amount of Rev1 in rev1flag, rev1flag pop2Δ, rev1flag pop1Δ and untagged rev1 strains. The lower panel shows Cdc2 as a loading control. B, Rev1 was coprecipitated with Pop1. rev1flag pop1V5 and rev1flag strains were grown, and whole cell extracts were prepared by the LiNi method. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. C, Rev1 was co-precipitated with Pop2. The rev1flag pop2V5 strain was grown, and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop2 input. The right panels show Rev1 and Pop2 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions. D, The Rev1dK mutant was not coprecipitated with Pop1. rev1flag pop1V5 and rev1dKflag pop1V5 strains were grown, and whole cell extracts were prepared. Immunoprecipitation was performed using anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panel shows Rev1 and Pop1 in anti-V5-immunoprecipitated fractions. E, Rev1KK (Rev1 761–818) associated with Pop1. Amino acids 761–818 of Rev1, which is the region deleted in Rev1dKK, were cloned into a pCAM1-flag expression vector. The pop1V5 strain with the pCAM1-rev1KKflag expression vector was grown at 30°C and whole cell extracts were prepared. Immunoprecipitation was performed using rabbit normal IgG or anti-V5 antibodies. The left panels show Rev1 and Pop1 input. The right panels show Rev1KK and Pop1 in rabbit normal IgG- or anti-V5-immunoprecipitated fractions.
Mentions: Next, we examined which ubiquitin ligase may be responsible for the degradation of Rev1. As shown in Fig 1C and 1D, Rev1 protein levels decreased dramatically at the onset of DNA replication. This pattern was similar to that of Cdc18, a functional homolog of budding yeast Cdc6 [49, 50]. The degradation of Cdc18 at G1/S depends on Pop1 and Pop2, subunits of SCF ubiquitin ligase [50, 51]. Therefore, we examined the involvement of Pop1 and Pop2 in the degradation of Rev1 at G1/S. Since pop1 and pop2 genes are not essential, we analyzed the protein levels of Rev1 in the pop1 or pop2 deletion background. The protein amount of Rev1 increased both in pop1Δ and pop2Δ mutants, although the contribution of pop1Δ was predominant (Fig 4A). Next, we examined the physical interactions between Rev1 and Pop1 or Pop2. As shown in Fig 4B and 4C, Rev1 was coprecipitated with Pop1 and Pop2. The interaction between Rev1 and Pop1 was also examined in cdc25-synchronized culture (S3 Fig). Rev1 was coprecipitated with Pop1, and the interaction peaked at about 100 min after the release. These data supported our hypothesis that Rev1 destruction at G1/S was controlled by SCF-dependent ubiquitination. To examine the contribution of the Lys-rich region of Rev1 for SCF-dependent proteolysis, we examined the protein-protein interactions between Pop1 and Rev1dK (Fig 4D). Interestingly, Rev1dK failed to interact with Pop1 following immunoprecipitation of Pop1. This result suggested that Rev1dK might become stable because it cannot interact with ubiquitin ligase. To test this hypothesis, we examined the interactions between Pop1 and Rev1KK (amino acids 761–818 of Rev1) (Fig 4E). Rev1KK was associated with Pop1. Based on these findings, we concluded that Rev1 destruction by the proteasome was mediated by Pop1 or Pop2 SCF ubiquitin ligase.

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