Limits...
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 stabilized under the restrictive condition for the mts2 or mts3 temperature-sensitive mutant.A, Protein level of Rev1 was increased after the temperature shift in the mts2 temperature-sensitive mutant. The mts2-U31 mutant, harboring-flag tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. Samples were collected every 2 h until 4 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1, Cdc13, Cdc2, and Pcn1. B, The protein level of Rev1dK did not increase at the restrictive temperature of mts2-U31. The panels show the protein expression of Rev1dK, Cdc13, and Cdc2 in the mts2-U31 strain, and that of Rev1dK and Cdc2 in the mts2wt control. The lanes represent the protein expression at 0, 2, and 4 h after the temperature shift in the mts2-U31 or mts2wt strain. C, The protein level of Rev1 increased after the temperature shift in the mts3 temperature-sensitive mutant. The mts3-U32 mutant, harboring flag-tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. The samples were collected every 2 h until 6 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1 and Cdc2, as well as CBB staining of the membrane. D, The protein level of Rev1dK did not increase at the restrictive temperature in the mts3-U32 strain. The panels show the protein expression of Rev1dK and Cdc2, as well as CBB staining of the membrane. The lanes represent the protein expression at 0, 2, 4, and 6 h after the temperature shift in the mts3-U32 strain.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493104&req=5

pone.0130000.g003: Rev1 protein was stabilized under the restrictive condition for the mts2 or mts3 temperature-sensitive mutant.A, Protein level of Rev1 was increased after the temperature shift in the mts2 temperature-sensitive mutant. The mts2-U31 mutant, harboring-flag tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. Samples were collected every 2 h until 4 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1, Cdc13, Cdc2, and Pcn1. B, The protein level of Rev1dK did not increase at the restrictive temperature of mts2-U31. The panels show the protein expression of Rev1dK, Cdc13, and Cdc2 in the mts2-U31 strain, and that of Rev1dK and Cdc2 in the mts2wt control. The lanes represent the protein expression at 0, 2, and 4 h after the temperature shift in the mts2-U31 or mts2wt strain. C, The protein level of Rev1 increased after the temperature shift in the mts3 temperature-sensitive mutant. The mts3-U32 mutant, harboring flag-tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. The samples were collected every 2 h until 6 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1 and Cdc2, as well as CBB staining of the membrane. D, The protein level of Rev1dK did not increase at the restrictive temperature in the mts3-U32 strain. The panels show the protein expression of Rev1dK and Cdc2, as well as CBB staining of the membrane. The lanes represent the protein expression at 0, 2, 4, and 6 h after the temperature shift in the mts3-U32 strain.

Mentions: Ubiquitin-dependent proteolysis occurs through the activity of proteasome complexes [44, 45]. Thus, we next analyzed Rev1 protein levels in proteasome-deficient conditions. First, we used random mutagenesis to construct the mts2/rpt2 temperature-sensitive mutant, which disrupted the function of a proteasomal subunit [46] following appropriate changes in temperature. The protein level of Rev1 was then examined at the restrictive condition of the temperature-sensitive mutant. The protein levels of Cdc13, which is controlled by proteasome-dependent proteolysis [47], and Rev1 were increased after mts2-U31 mutant cells were arrested (Fig 3A). In contrast, the protein levels of the Rev1dK mutant did not increase after mts2-U31-dependent cell cycle arrest (Fig 3B). The apparent decrease in the protein level of Rev1dK resulted from the increasing temperature and cell cycle arrest in M phase caused by mts2-U31. To further confirm the contribution of the proteasome to Rev1 protein levels, we also constructed mts3/rpn12 [48] temperature-sensitive mutants. Similarly, Rev1 protein levels increased in mts3-U32 cells at the restrictive temperature (Fig 3C), and the amount of Rev1dK did not increase under the same conditions (Fig 3D). These data clearly indicated that Rev1 protein levels were controlled by proteasomal degradation.


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 stabilized under the restrictive condition for the mts2 or mts3 temperature-sensitive mutant.A, Protein level of Rev1 was increased after the temperature shift in the mts2 temperature-sensitive mutant. The mts2-U31 mutant, harboring-flag tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. Samples were collected every 2 h until 4 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1, Cdc13, Cdc2, and Pcn1. B, The protein level of Rev1dK did not increase at the restrictive temperature of mts2-U31. The panels show the protein expression of Rev1dK, Cdc13, and Cdc2 in the mts2-U31 strain, and that of Rev1dK and Cdc2 in the mts2wt control. The lanes represent the protein expression at 0, 2, and 4 h after the temperature shift in the mts2-U31 or mts2wt strain. C, The protein level of Rev1 increased after the temperature shift in the mts3 temperature-sensitive mutant. The mts3-U32 mutant, harboring flag-tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. The samples were collected every 2 h until 6 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1 and Cdc2, as well as CBB staining of the membrane. D, The protein level of Rev1dK did not increase at the restrictive temperature in the mts3-U32 strain. The panels show the protein expression of Rev1dK and Cdc2, as well as CBB staining of the membrane. The lanes represent the protein expression at 0, 2, 4, and 6 h after the temperature shift in the mts3-U32 strain.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493104&req=5

pone.0130000.g003: Rev1 protein was stabilized under the restrictive condition for the mts2 or mts3 temperature-sensitive mutant.A, Protein level of Rev1 was increased after the temperature shift in the mts2 temperature-sensitive mutant. The mts2-U31 mutant, harboring-flag tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. Samples were collected every 2 h until 4 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1, Cdc13, Cdc2, and Pcn1. B, The protein level of Rev1dK did not increase at the restrictive temperature of mts2-U31. The panels show the protein expression of Rev1dK, Cdc13, and Cdc2 in the mts2-U31 strain, and that of Rev1dK and Cdc2 in the mts2wt control. The lanes represent the protein expression at 0, 2, and 4 h after the temperature shift in the mts2-U31 or mts2wt strain. C, The protein level of Rev1 increased after the temperature shift in the mts3 temperature-sensitive mutant. The mts3-U32 mutant, harboring flag-tagged Rev1, was first grown at 25°C, and the temperature was then shifted to 36.5°C. The samples were collected every 2 h until 6 h after the shift. Whole cell extracts were prepared. The panels show the protein expression of Rev1 and Cdc2, as well as CBB staining of the membrane. D, The protein level of Rev1dK did not increase at the restrictive temperature in the mts3-U32 strain. The panels show the protein expression of Rev1dK and Cdc2, as well as CBB staining of the membrane. The lanes represent the protein expression at 0, 2, 4, and 6 h after the temperature shift in the mts3-U32 strain.
Mentions: Ubiquitin-dependent proteolysis occurs through the activity of proteasome complexes [44, 45]. Thus, we next analyzed Rev1 protein levels in proteasome-deficient conditions. First, we used random mutagenesis to construct the mts2/rpt2 temperature-sensitive mutant, which disrupted the function of a proteasomal subunit [46] following appropriate changes in temperature. The protein level of Rev1 was then examined at the restrictive condition of the temperature-sensitive mutant. The protein levels of Cdc13, which is controlled by proteasome-dependent proteolysis [47], and Rev1 were increased after mts2-U31 mutant cells were arrested (Fig 3A). In contrast, the protein levels of the Rev1dK mutant did not increase after mts2-U31-dependent cell cycle arrest (Fig 3B). The apparent decrease in the protein level of Rev1dK resulted from the increasing temperature and cell cycle arrest in M phase caused by mts2-U31. To further confirm the contribution of the proteasome to Rev1 protein levels, we also constructed mts3/rpn12 [48] temperature-sensitive mutants. Similarly, Rev1 protein levels increased in mts3-U32 cells at the restrictive temperature (Fig 3C), and the amount of Rev1dK did not increase under the same conditions (Fig 3D). These data clearly indicated that Rev1 protein levels were controlled by proteasomal degradation.

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