Increased expression of Rev1 sensitized the cells to cisplatin.

<p><b>A,</b><i>rev1dK</i> exhibited cisplatin (CDDP) sensitivity. wt, <i>rev1Δ</i>, and <i>rev1dK</i> 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, <i>rev1Δ</i>, and <i>rev1dK</i> strains on cisplatin minus control and on a YES plate containing 100 μM cisplatin. <b>B,</b> Overexpression of <i>rev1</i> conferred sensitivity to cisplatin. Cells harboring pREP41, pREP41-<i>rev1</i>, or pREP41-<i>rev1dK</i> 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.</p

Eso1 and Rev1 were associated in G₁ phase.

<p><b>A,</b><i>eso1^Δpolh</i> and <i>rev1Δ</i> 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/m<i>²</i>). Plates were incubated at 30°C, and the growth of wt, <i>eso1^Δpolh</i>, and <i>rev1Δ</i> strains was observed 3 days after the irradiation. <b>B,</b> Eso1 associated with Rev1 in <i>cdc10</i>-arrested extracts. <i>cdc10 rev1^flag eso1^V5</i> and <i>cdc25 rev1^flag eso1^V5</i> 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 <i>cdc10</i> strain at 25°C, the <i>cdc10</i> strain at 36.5°C, the <i>cdc25</i> strain at 25°C, and the <i>cdc25</i> strain at 36.5°C. <b>C,</b> Eso1 was coprecipitated with Rev7. <i>eso1^myc</i> and <i>eso1^myc rev7^flag</i> 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. <b>D,</b> Rev7 failed to coprecipitate Eso1 in the absence of Rev1. <i>eso1^myc rev7^flag</i> and <i>eso1^myc rev7^flag rev1Δ</i> 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.</p

Rev1 protein was stabilized under the restrictive condition for the <i>mts2</i> or <i>mts3</i> temperature-sensitive mutant.

<p><b>A,</b> Protein level of Rev1 was increased after the temperature shift in the <i>mts2</i> temperature-sensitive mutant. The <i>mts2-U31</i> 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>B,</b> The protein level of Rev1dK did not increase at the restrictive temperature of <i>mts2-U31</i>. The panels show the protein expression of Rev1dK, Cdc13, and Cdc2 in the <i>mts2-U31</i> strain, and that of Rev1dK and Cdc2 in the <i>mts2^wt</i> control. The lanes represent the protein expression at 0, 2, and 4 h after the temperature shift in the <i>mts2-U31 or mts2^wt</i> strain. <b>C,</b> The protein level of Rev1 increased after the temperature shift in the <i>mts3</i> temperature-sensitive mutant. The <i>mts3-U32</i> 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. <b>D,</b> The protein level of Rev1dK did not increase at the restrictive temperature in the <i>mts3-U32</i> 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 <i>mts3-U32</i> strain.</p

Rev1 protein was most abundant in G¹ phase.

<p><b>A,</b> The TLS polymerases Eso1 and Kpa1 were upregulated in S phase. Eso1, Kpa1, and Rev7 protein expression levels were examined after <i>cdc25</i>-dependent block and release. Whole cell extracts were prepared by the boiling method, and western blotting was performed at different times (20–200 min) after the release. The graph represents the septation index after release. The lower panels show the expression patterns of Eso1, Kpa1, Rev7, Pcn1, Cdc13 (cyclin B), and Cdc2. <b>B,</b> Rev3 protein expression peaked during S phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The middle panel shows the expression pattern of Rev3, which was detected by anti-flag antibodies. The lower panel shows the protein expression of Pcn1 in the chromatin fraction. <b>C,</b> The protein amount of Rev1 was highest in G₁ phase. Whole cell extracts were prepared by the boiling method. The graph represents the septation index after release. The panels show the expression patterns of Rev1, Cdc13, and Cdc2. <b>D,</b> The protein expression of Rev1 in cell cycle mutants. <i>cdc10</i>, <i>cdc20</i>, <i>cdc22</i>, <i>cdc21</i>, <i>cdc17</i>, and <i>cdc25</i> mutants harboring flag-tagged <i>rev1</i> were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the boiling method. The amount of Rev1 was analyzed. <b>E,</b> Modified forms of the Rev1 protein were observed only in <i>cdc20</i> or <i>cdc22</i>. <i>cdc10</i>, <i>cdc20</i>, <i>cdc22</i>, and <i>cdc25</i> cells harboring flag-tagged <i>rev1</i> were arrested at 36.5°C for 4 h. Whole cell extracts were prepared by the LiNi method and then subjected to immunoprecipitation. Rev1 protein expression in each sample was roughly adjusted, and western blotting was performed.</p

An internal deletion mutant stabilized Rev1 protein in the S phase.

<p><b>A,</b> 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>B,</b> The Rev1dKK mutant stabilized Rev1 protein. Flag-tagged <i>rev1^wt</i> and <i>rev1dKK</i> 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 Rev1<i>^wt</i>, and the right lane represents Rev1dKK. <b>C,</b> Rev1dKK protein was stable in S phase. Time course samples of the <i>rev1dKK^flag cdc25</i> 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.</p

Pop1 and Pop2 were responsible for the stability of Rev1.

<p><b>A,</b><i>pop1</i> and <i>pop2</i> deletion mutants stabilized Rev1 protein. wt, <i>pop1Δ</i>, and <i>pop2Δ</i> strains harboring flag-tagged <i>rev1</i> and an untagged <i>rev1</i> 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 <i>rev1^flag</i>, <i>rev1^flag pop2Δ</i>, <i>rev1^flag pop1Δ</i> and untagged <i>rev1</i> strains. The lower panel shows Cdc2 as a loading control. <b>B,</b> Rev1 was coprecipitated with Pop1. <i>rev1^flag pop1^V5</i> and <i>rev1^flag</i> 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. <b>C,</b> Rev1 was co-precipitated with Pop2. The <i>rev1^flag pop2^V5</i> 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. <b>D,</b> The Rev1dK mutant was not coprecipitated with Pop1. <i>rev1^flag pop1^V5</i> and <i>rev1dK^flag pop1^V5</i> 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. <b>E,</b> 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 <i>pop1^V5</i> strain with the pCAM1-<i>rev1KKflag</i> 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.</p

The protein expression of Rev1 was upregulated in response to DNA damage.

<p><b>A,</b> Rev1 protein expression after mutagen treatment. At the logarithmic growth phase, wt cells harboring flag-tagged <i>rev1</i> were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested. Whole cell extracts were prepared by the boiling method, and protein levels were examined by western blotting. The upper panel represents flag-tagged Rev1, and the lower panel shows CBB staining of the membrane. <b>B,</b> The upregulation of Rev1 was dependent on Rad3. At the logarithmic growth phase, <i>rev1^flag</i> and <i>rev1^flag rad3Δ</i> strains were treated with 0, 1, 5, 10, or 25 μM cisplatin for 3 h. Cells were then harvested, and whole cell extracts were prepared by the boiling method. Extracts were then subjected to western blotting. The upper panels show Rev1 and CBB staining of the <i>rev1^flag</i> strain, and the lower panels show those of the <i>rev1^flag rad3Δ</i> strain. <b>C,</b> The promoter region was important for the upregulation of Rev1 after DNA damage. At the logarithmic growth phase, <i>cam1-rev1</i> cells were treated with no drug (-), 50 μM cisplatin (CDDP), 10 mM hydroxyurea (HU), 0.008% MMS, 500 nM 4NQO, or 40 μM camptothecin (CPT). After a 4-h incubation, cells were harvested, and whole cell extracts were prepared by the boiling method. Protein expression was then examined by western blotting. The upper panel shows Rev1, and the lower panel shows CBB staining.</p

A model for the protein level regulation of Rev1 and TLS.

<p>In G1 phase, Rev1 is abundant and Rev1-dependent loading of TLS polymerase may occur. At the onset of S phase, Rev1 is destroyed in a SCF-dependent manner and chromatin-loaded Eso1/polη serves as an initiator of TLS. When DNA is damaged, the DNA structure checkpoint increases the protein level of Rev1 and facilitates polymerase switching among TLS polymerases.</p