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. 2016 Feb 26;291(9):4442-52.
doi: 10.1074/jbc.M115.671057. Epub 2016 Jan 6.

The Budding Yeast Ubiquitin Protease Ubp7 Is a Novel Component Involved in S Phase Progression

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Free PMC article

The Budding Yeast Ubiquitin Protease Ubp7 Is a Novel Component Involved in S Phase Progression

Stefanie Böhm et al. J Biol Chem. .
Free PMC article

Abstract

DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7.

Keywords: DNA damage response; DNA replication; deubiquitination; deubiquitylation (deubiquitination); molecular evolution; yeast genetics.

Figures

FIGURE 1.
FIGURE 1.
Ubp7 is linked to the DNA damage response. a, ERC values between the Ubp7 and DNA repair pathways show statistically significant correlations with HR and NER but not with base excision repair (BER), mismatch repair (MMR), nonhomologous end joining (NHEJ), or endocytosis (Endocyt). Violin plots show the distribution of ERC values between the UBP7 and DNA repair pathways. Curves show a smoothed representation of their density, whereas tick marks show the exact values. p values reflect the significance of elevation above the genome-wide distribution. b, 5-fold serial dilutions of wild-type, ubp7Δ, and rad51Δ (control) strains grown on YPD, YPD with 150 mm HU, YPD with 0.02% MMS, or YPD and irradiated with 100 Gy of γ radiation (IR). c, 5-fold serial dilutions of the same strains as in a grown on SC with 0.03% DMSO or the indicated cisplatin (in DMSO) concentrations. d, WT, ubp7Δ, and rad51Δ cells were measured for cell viability by plating on YPD medium containing the indicated doses of HU and compared with their corresponding untreated YPD control. e, wild-type, ubp7Δ, and rad51Δ cultures were grown in SC medium and plated on SC plates containing the indicated concentrations of HU. Plates were imaged 24 h after plating.
FIGURE 2.
FIGURE 2.
Ubp7 is not an NER or HR component. a, 5-fold serial dilutions of the indicated strains on YPD plates exposed to 0, 50, or 100 J/m2 UV radiation. b, 5-fold serial dilutions of the strains as in a, spotted on SC plates with DMSO or the indicated cisplatin concentrations. c, comparison of total recombination rates in a wild-type and ubp7Δ strain chromosomally harboring the direct repeat recombination assay. Cells were grown in SC medium or SC medium containing DMSO as a control compared with cells exposed to 35 mm HU or 10 μg/ml cisplatin for 26 h and then plated. The graph depicts the mean recombination rate from four to five independent trials ± S.D. The recombination rate in WT and ubp7Δ cells is not significantly different by Student's t test for each condition tested. d, spontaneous CAN1 mutation frequency in wild-type, ubp7Δ, rad51Δ, and ubp7Δ rad51Δ. The graph depicts the mean recombination frequency from three independent trials ± S.D. Only the difference between rad51Δ and the wild-type is statistically significant by Student's t test (*, p < 0.05). e, genetic analysis with the HR genes RAD51 and SGS1. 5-Fold serial dilutions were spotted on SC plates containing 25 and 100 mm HU and incubated at 30 °C for 3 days. f, genetic analysis of ubp7Δ with a deletion of MUS81. Serial dilutions of the indicated strains were performed as in a.
FIGURE 3.
FIGURE 3.
S phase progression is altered in ubp7 cells. a, live-cell microscopy of wild-type and ubp7Δ Rfa1-YFP cells left untreated or after 2 h with 100 mm HU. A representative z plane of differential interference contrast (DIC)/YFP for each strain is shown. Arrowheads mark Rfa1 foci. b, quantification of a. The graph depicts the mean percentage of cells with an Rfa1-YFP focus ± S.E.M. (n (WT, untreated) = 1309, n (ubp7Δ, untreated) = 1359, n (WT, HU) = 1007, n (ubp7Δ, HU) = 1059). The indicated p values were calculated by Fischer's exact test. n.s., not significant. c, FACS analysis of WT and ubp7Δ cells in log phase (log), after α factor arrest (aF), and after release from 4 h of treatment with 200 mm HU (10, 20, 30, 40, 50, and 60 min). d, two-dimensional gel electrophoresis analysis with a probe recognizing the early origin of replication, ARS305, following HU treatment of α factor-arrested cells. The arrowhead indicates where bubbles are accumulating in ubp7Δ cells.
FIGURE 4.
FIGURE 4.
Epistasis analysis of UBP7 with intra-S phase checkpoint genes. a, schematic of relevant factors for Mec1 and Rad53 activation. b, 5-fold serial dilutions of the indicated strains spotted on plates containing HU and incubated at 30 °C for 3 d. c, Mrc1 protein levels and phosphorylation status in wild-type and ubp7Δ MRC1–9myc strains. Cultures were left untreated or exposed to 100 mm HU or 0.3% MMS for 2 h before whole-cell lysates were prepared. Shown is a Western blotting analysis against myc (Mrc1) and Adh1 (loading).
FIGURE 5.
FIGURE 5.
Deletion of UBP7 affects H2B ubiquitination. a, ERC values between Ubp7 and histone modification genes are elevated significantly compared with the genome-wide distribution. The violin plot shows the actual ERC values (tick marks) and their smoothed densities. b, genetic interaction of ubp7Δ with the histone chaperone ASF1 and histone-modifying E2 RAD6. Shown are 5-fold serial dilutions of the indicated strains on plates containing 1 and 25 mm HU, respectively. c, chromatin-binding assay for Ubp7–3HA. Shown are Western blotting analyses of whole-cell extract (W), supernatant (S), and chromatin-bound (C) fractions from asynchronous cultures (as) or those synchronized in G1 (alpha), S (HU), and G2/M phase (noc). Antibodies against HA, Clb2, GAPDH, and H2B were used. d, genetic interaction of ubp7Δ with htb-K123R. Shown are 5-fold serial dilutions of the indicated strains on plates containing the respective amounts of HU. Note that the strains used in this figure were in the FY2 genetic background, and, therefore, we observed a modest variation in the DNA damage sensitivity of ubp7Δ cells. e, steady-state H2B ubiquitination at Lys-123 in untreated asynchronous wild-type and ubp7Δ cultures. Shown is a Western blotting analysis for ubiquitinated H2B and total H2B. f, quantification of e by determining the ratio of H2B-Ub to total H2B and normalizing to the wild type. The graph represents the mean of four independent trials ± S.D.
FIGURE 6.
FIGURE 6.
Schematic of the effect of UBP7 deletion. During DNA replication in a wild-type cell, a small amount of single-stranded DNA is exposed by the replicative helicase, and this single-stranded DNA is coated rapidly by RPA. The histone chaperone Asf1 deposits nucleosomes onto the newly synthesized DNA, and Rad6-Bre1 is responsible for H2B monoubiquitination at Lys-123. In a ubp7Δ cell, the replication fork is not stabilized properly, and this leads to HU and cisplatin sensitivity and elevated H2B ubiquitination. If an ubp7Δ strain is treated with HU, then there is an increase in single-stranded DNA coated by RPA and a requirement for SGS1, MRC1, and MUS81 for viability.

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