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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

Increased expression of Rev1 sensitized the cells to cisplatin.A,rev1dK exhibited cisplatin (CDDP) sensitivity. wt, rev1Δ, and rev1dK strains were grown. Cells were spotted on YES plates containing cisplatin with 5-fold serial dilutions, and the plates were incubated at 30°C for 3 days. The panels represent the growth of wt, rev1Δ, and rev1dK strains on cisplatin minus control and on a YES plate containing 100 μM cisplatin. B, Overexpression of rev1 conferred sensitivity to cisplatin. Cells harboring pREP41, pREP41-rev1, or pREP41-rev1dK plasmids were grown in EMM media without thiamine and spotted on an EMM plate containing 0 or 25μM CDDP with 5-fold serial dilutions. The plates were incubated at 30°C for 3 days. The panels represent the growth on an EMM plate without CDDP or containing 25 μM CDDP.
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pone.0130000.g006: Increased expression of Rev1 sensitized the cells to cisplatin.A,rev1dK exhibited cisplatin (CDDP) sensitivity. wt, rev1Δ, and rev1dK strains were grown. Cells were spotted on YES plates containing cisplatin with 5-fold serial dilutions, and the plates were incubated at 30°C for 3 days. The panels represent the growth of wt, rev1Δ, and rev1dK strains on cisplatin minus control and on a YES plate containing 100 μM cisplatin. B, Overexpression of rev1 conferred sensitivity to cisplatin. Cells harboring pREP41, pREP41-rev1, or pREP41-rev1dK plasmids were grown in EMM media without thiamine and spotted on an EMM plate containing 0 or 25μM CDDP with 5-fold serial dilutions. The plates were incubated at 30°C for 3 days. The panels represent the growth on an EMM plate without CDDP or containing 25 μM CDDP.

Mentions: As shown in the previous section, Rev1 may serve as a regulator of TLS. However, it is unclear why Rev1 protein, which was abundantly expressed in G1 phase, must be destroyed when cells progress into the S phase. Indeed, Rev1 destruction may have positive effects on TLS as overproduction of the C-term domain of Rev1 has been reported to sensitize budding yeast to DNA damage [56]. To examine this possibility, we decided to analyze the DNA damage sensitivity of rev1dK, a proteasome-insensitive mutant. The rev1 deletion mutant exhibited sensitivities to various DNA-damaging agents (S7 Fig). Among the agents we tested, rev1Δ exhibited the highest sensitivity to cisplatin (CDDP); therefore, we utilized cisplatin as a damaging agent. Although weaker than that of rev1Δ, rev1dK also exhibited cisplatin sensitivity (Fig 6A). Interestingly, as shown in S2, S4, and S5 Figs, the rev1dK structure maintained all known functional domains of Rev1 intact; thus, it is highly possible that the cisplatin sensitivity of the rev1dK mutant was conferred by the increased amount of Rev1. To investigate this possibility, we examined the effects of Rev1 protein levels on cisplatin sensitivity by overexpressing Rev1. rev1wt and rev1dK were overexpressed under the thiamine-repressive nmt41 promoter under activation conditions. To confirm the overproduction of Rev1, we also overexpressed a flag-tagged version of rev1 and the expression level was examined (S6 Fig). Interestingly, both rev1dK and rev1wt overproduction enhanced the cellular sensitivity to cisplatin (Fig 6B), indicating that Rev1 was able to serve as a negative regulator for TLS.


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)

Increased expression of Rev1 sensitized the cells to cisplatin.A,rev1dK exhibited cisplatin (CDDP) sensitivity. wt, rev1Δ, and rev1dK strains were grown. Cells were spotted on YES plates containing cisplatin with 5-fold serial dilutions, and the plates were incubated at 30°C for 3 days. The panels represent the growth of wt, rev1Δ, and rev1dK strains on cisplatin minus control and on a YES plate containing 100 μM cisplatin. B, Overexpression of rev1 conferred sensitivity to cisplatin. Cells harboring pREP41, pREP41-rev1, or pREP41-rev1dK plasmids were grown in EMM media without thiamine and spotted on an EMM plate containing 0 or 25μM CDDP with 5-fold serial dilutions. The plates were incubated at 30°C for 3 days. The panels represent the growth on an EMM plate without CDDP or containing 25 μM CDDP.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130000.g006: Increased expression of Rev1 sensitized the cells to cisplatin.A,rev1dK exhibited cisplatin (CDDP) sensitivity. wt, rev1Δ, and rev1dK strains were grown. Cells were spotted on YES plates containing cisplatin with 5-fold serial dilutions, and the plates were incubated at 30°C for 3 days. The panels represent the growth of wt, rev1Δ, and rev1dK strains on cisplatin minus control and on a YES plate containing 100 μM cisplatin. B, Overexpression of rev1 conferred sensitivity to cisplatin. Cells harboring pREP41, pREP41-rev1, or pREP41-rev1dK plasmids were grown in EMM media without thiamine and spotted on an EMM plate containing 0 or 25μM CDDP with 5-fold serial dilutions. The plates were incubated at 30°C for 3 days. The panels represent the growth on an EMM plate without CDDP or containing 25 μM CDDP.
Mentions: As shown in the previous section, Rev1 may serve as a regulator of TLS. However, it is unclear why Rev1 protein, which was abundantly expressed in G1 phase, must be destroyed when cells progress into the S phase. Indeed, Rev1 destruction may have positive effects on TLS as overproduction of the C-term domain of Rev1 has been reported to sensitize budding yeast to DNA damage [56]. To examine this possibility, we decided to analyze the DNA damage sensitivity of rev1dK, a proteasome-insensitive mutant. The rev1 deletion mutant exhibited sensitivities to various DNA-damaging agents (S7 Fig). Among the agents we tested, rev1Δ exhibited the highest sensitivity to cisplatin (CDDP); therefore, we utilized cisplatin as a damaging agent. Although weaker than that of rev1Δ, rev1dK also exhibited cisplatin sensitivity (Fig 6A). Interestingly, as shown in S2, S4, and S5 Figs, the rev1dK structure maintained all known functional domains of Rev1 intact; thus, it is highly possible that the cisplatin sensitivity of the rev1dK mutant was conferred by the increased amount of Rev1. To investigate this possibility, we examined the effects of Rev1 protein levels on cisplatin sensitivity by overexpressing Rev1. rev1wt and rev1dK were overexpressed under the thiamine-repressive nmt41 promoter under activation conditions. To confirm the overproduction of Rev1, we also overexpressed a flag-tagged version of rev1 and the expression level was examined (S6 Fig). Interestingly, both rev1dK and rev1wt overproduction enhanced the cellular sensitivity to cisplatin (Fig 6B), indicating that Rev1 was able to serve as a negative regulator for TLS.

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