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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

An internal deletion mutant stabilized Rev1 protein in the S phase.A, Schematic diagram of the fission yeast Rev1 domain structure. Parallelogram, BRCT; rectangle, Y family-conserved region; ellipse, ubiquitin binding motif; hexagon, Lys-rich region. Rev1dK and Rev1dKK are internal deletion mutants lacking the amino acids from 761 to 818 and 761 to 797, respectively. B, The Rev1dKK mutant stabilized Rev1 protein. Flag-tagged rev1wt and rev1dKK cells were grown, and whole cell extracts were prepared by the boiling method. The upper panel shows a western blot of Rev1 protein, and the lower panel shows CBB staining of the membrane as a loading control. The left lane represents Rev1wt, and the right lane represents Rev1dKK. C, Rev1dKK protein was stable in S phase. Time course samples of the rev1dKKflag cdc25 strain were prepared. The samples were taken every 30 min after the release. The lower panels show the expression patterns of Rev1dKK, Cdc13, and Cdc2.
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pone.0130000.g002: An internal deletion mutant stabilized Rev1 protein in the S phase.A, Schematic diagram of the fission yeast Rev1 domain structure. Parallelogram, BRCT; rectangle, Y family-conserved region; ellipse, ubiquitin binding motif; hexagon, Lys-rich region. Rev1dK and Rev1dKK are internal deletion mutants lacking the amino acids from 761 to 818 and 761 to 797, respectively. B, The Rev1dKK mutant stabilized Rev1 protein. Flag-tagged rev1wt and rev1dKK cells were grown, and whole cell extracts were prepared by the boiling method. The upper panel shows a western blot of Rev1 protein, and the lower panel shows CBB staining of the membrane as a loading control. The left lane represents Rev1wt, and the right lane represents Rev1dKK. C, Rev1dKK protein was stable in S phase. Time course samples of the rev1dKKflag cdc25 strain were prepared. The samples were taken every 30 min after the release. The lower panels show the expression patterns of Rev1dKK, Cdc13, and Cdc2.

Mentions: We found that Rev1 protein levels dropped sharply during G1/S phase. Interestingly, a similar destabilization of Rev1 at G1/S was observed in budding yeast, and this destabilization was found to be dependent on the proteasome [42]. Therefore, the proteasome may also control Rev1 protein levels in fission yeast. Analysis of Rev1 protein levels (Fig 1C) demonstrated that the band representing Rev1 protein during G1/S was often present as a high-molecular-weight smear on western blotting, also supporting the potential proteasome-dependent regulation of Rev1. To examine the nature of this high-molecular-weight band, we immunoprecipitated Rev1 in cdc10-, cdc20-, cdc22-, and cdc25-arrested cultures. When the total amount of Rev1 was roughly equalized, the high-molecular-weight bands (resembling a ladder) were observed only in cdc20- and cdc22-arrested extracts (Fig 1E), suggesting that Rev1 underwent ubiquitin-dependent proteolysis. Ubiquitination targets the lysine residues of the protein; thus, we first constructed Rev1dKK, an internal deletion mutant (Δ761–818) that lacked the lysine-rich region near the C terminus (Fig 2A). As expected, Rev1dKK protein levels were much higher than those of Rev1wt, even in unsynchronized conditions (Fig 2B). Next, we examined the protein level of Rev1 in the Rev1dKK mutant in cdc25-synchronized cultures. As shown in Fig 2C, Rev1dKK protein levels peaked at S phase instead of G1 phase. Because cell cycle-dependent oscillation of rev1 mRNA was previously reported [43], the observed protein level oscillation of Rev1dKK probably resulted from regulation of the mRNA level. We also created a Rev1dK internal deletion mutant (Δ761–797) because the Rev1dKK mutant lacked a ubiquitin-binding motif, which is important for the function of Rev1. Rev1 protein was also elevated in the Rev1dK mutant compared to the Rev1wt strain (S2 Fig). These data suggested that Rev1 underwent ubiquitin-dependent proteolysis at G1/S phase.


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)

An internal deletion mutant stabilized Rev1 protein in the S phase.A, Schematic diagram of the fission yeast Rev1 domain structure. Parallelogram, BRCT; rectangle, Y family-conserved region; ellipse, ubiquitin binding motif; hexagon, Lys-rich region. Rev1dK and Rev1dKK are internal deletion mutants lacking the amino acids from 761 to 818 and 761 to 797, respectively. B, The Rev1dKK mutant stabilized Rev1 protein. Flag-tagged rev1wt and rev1dKK cells were grown, and whole cell extracts were prepared by the boiling method. The upper panel shows a western blot of Rev1 protein, and the lower panel shows CBB staining of the membrane as a loading control. The left lane represents Rev1wt, and the right lane represents Rev1dKK. C, Rev1dKK protein was stable in S phase. Time course samples of the rev1dKKflag cdc25 strain were prepared. The samples were taken every 30 min after the release. The lower panels show the expression patterns of Rev1dKK, Cdc13, and Cdc2.
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Related In: Results  -  Collection

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pone.0130000.g002: An internal deletion mutant stabilized Rev1 protein in the S phase.A, Schematic diagram of the fission yeast Rev1 domain structure. Parallelogram, BRCT; rectangle, Y family-conserved region; ellipse, ubiquitin binding motif; hexagon, Lys-rich region. Rev1dK and Rev1dKK are internal deletion mutants lacking the amino acids from 761 to 818 and 761 to 797, respectively. B, The Rev1dKK mutant stabilized Rev1 protein. Flag-tagged rev1wt and rev1dKK cells were grown, and whole cell extracts were prepared by the boiling method. The upper panel shows a western blot of Rev1 protein, and the lower panel shows CBB staining of the membrane as a loading control. The left lane represents Rev1wt, and the right lane represents Rev1dKK. C, Rev1dKK protein was stable in S phase. Time course samples of the rev1dKKflag cdc25 strain were prepared. The samples were taken every 30 min after the release. The lower panels show the expression patterns of Rev1dKK, Cdc13, and Cdc2.
Mentions: We found that Rev1 protein levels dropped sharply during G1/S phase. Interestingly, a similar destabilization of Rev1 at G1/S was observed in budding yeast, and this destabilization was found to be dependent on the proteasome [42]. Therefore, the proteasome may also control Rev1 protein levels in fission yeast. Analysis of Rev1 protein levels (Fig 1C) demonstrated that the band representing Rev1 protein during G1/S was often present as a high-molecular-weight smear on western blotting, also supporting the potential proteasome-dependent regulation of Rev1. To examine the nature of this high-molecular-weight band, we immunoprecipitated Rev1 in cdc10-, cdc20-, cdc22-, and cdc25-arrested cultures. When the total amount of Rev1 was roughly equalized, the high-molecular-weight bands (resembling a ladder) were observed only in cdc20- and cdc22-arrested extracts (Fig 1E), suggesting that Rev1 underwent ubiquitin-dependent proteolysis. Ubiquitination targets the lysine residues of the protein; thus, we first constructed Rev1dKK, an internal deletion mutant (Δ761–818) that lacked the lysine-rich region near the C terminus (Fig 2A). As expected, Rev1dKK protein levels were much higher than those of Rev1wt, even in unsynchronized conditions (Fig 2B). Next, we examined the protein level of Rev1 in the Rev1dKK mutant in cdc25-synchronized cultures. As shown in Fig 2C, Rev1dKK protein levels peaked at S phase instead of G1 phase. Because cell cycle-dependent oscillation of rev1 mRNA was previously reported [43], the observed protein level oscillation of Rev1dKK probably resulted from regulation of the mRNA level. We also created a Rev1dK internal deletion mutant (Δ761–797) because the Rev1dKK mutant lacked a ubiquitin-binding motif, which is important for the function of Rev1. Rev1 protein was also elevated in the Rev1dK mutant compared to the Rev1wt strain (S2 Fig). These data suggested that Rev1 underwent ubiquitin-dependent proteolysis at G1/S phase.

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