PCNA-Dependent Cleavage and Degradation of SDE2 Regulates Response to Replication Stress

<div><p>Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4^CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4^CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function.</p></div

Degradation of cleaved C-terminal SDE2 is regulated by chromatin association and CRL4^CDT2.

<p><b>(A)</b> HeLa cells expressing full-length GFP-SDE2 were left untreated or treated with 40 J/m² UVC for 4 h and fractionated into cytosolic/nucleoplasmic (S) vs. chromatin-enriched (P) fractions. The level of cleaved C-terminal SDE2 (C-SDE2) was analyzed by Western blotting. <b>(B)</b> (left) A schematic of the C-terminal Flag-tagged SDE2 ΔSAP mutant (aa 1–394). (right) HeLa cells expressing SDE2-Flag wild-type or ΔSAP mutant were treated with 40 J/m² UVC for 4 h, fractionated, and analyzed by Western blotting. <b>(C)</b> HeLa cells expressing full-length GFP-SDE2 variants were irradiated with UVC and fractionated for Western blot analysis. <b>(D)</b> HeLa cells transfected with siRNA control or CDT2 for 48 h were treated with 40 J/m² UVC for 4 h and fractionated. Endogenous cleaved SDE2 levels were analyzed by Western blotting. <b>(E)</b> 293T cells were transiently expressed with HA-CDT2 along with Flag-SDE2 variants, treated with 10 μM MG132 for 4h, and cell lysates were subjected to anti-Flag IP and Western blotting. <b>(F)</b> HeLa cells transfected with HA-CDT2 plasmid were fractionated to S and P fractions, and endogenous C-SDE2 levels were analyzed by Western blotting. <b>(G)</b> Immunoblots of the <i>in vivo</i> ubiquitination assay in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006465#pgen.1006465.g003" target="_blank">Fig 3G</a> were reprobed to visualize the polyubiquitin conjugates of C-SDE2.</p

Failure to degrade SDE2 impairs S phase progression and cellular survival.

<p><b>(A)</b> (left) A total of 5 x 10³ HeLa cells transiently expressing GFP-SDE2 variants were plated, and cell numbers were counted at the indicated times. Data shown are the mean ± SD of three independent experiments. * <i>p</i> < 0.01 GA and PIP compared with vector or wild-type. (top) The expression of SDE2 variants was analyzed by Western blotting. (EV: empty vector) <b>(B)</b> A schematic for monitoring S phase progression. HeLa cells were incubated with 2 mM HU for 18 h, pulsed with 10 μM BrdU for 30 min, and released into fresh medium. A representative flow cytometry gating of BrdU⁺ cells that traverse from G1/S boundary (R2) to early (R3) and late (R4) S phases is shown. <b>(C)</b> HeLa cells expressing vector control, wild-type, GA, or PIP SDE2-Flag variants were analyzed as (B) following release from HU. Data shown are the mean ± SD of two independent experiments. * <i>p</i> < 0.01 for R2 cells compared with EV. ** <i>p</i> < 0.01 R3 cells compared with EV. n.s.: not significant compared with EV. <b>(D)</b> (left) A total of 5 x 10³ HeLa cells stably expressing vector, SDE2-Flag PIP or PIP with ΔSAP mutant were plated, and cell numbers were counted at the indicated times. Data shown are the mean ± SD of three independent experiments. * <i>p</i> < 0.01 compared with empty vector or PIP. (right) Expression of SDE2 variants was analyzed by Western blotting. <b>(E)</b> HeLa cells transiently expressing GFP-SDE2 variants were plated to 96 well plates, treated with indicated doses of HU, and cell viability was determined by CellTiter-Glo luminescence. Data shown are the mean ± SD of three independent experiments. * <i>p</i> < 0.01 for GA and PIP compared to empty vector.</p

SDE2 suppresses UVC-inducible PCNA monoubiquitination.

<p><b>(A-B)</b> Western blot analyses of siRNA-transfected HeLa cells irradiated with 40 J/m² UVC for the indicated times. <b>(C)</b> HeLa cells were sequentially transfected with SDE2 siRNA and siRNA-resistant GFP-SDE2 cDNA (GFP-SDE2*), UVC irradiated, and analyzed by Western blotting. <b>(D)</b> HeLa cells transfected with SDE2 siRNA were transiently expressed with siRNA-resistant GFP-SDE2 wild-type, ΔSDE2, or GA mutant, UVC irradiated, and analyzed by Western blotting. <b>(E)</b> Expression of cleaved C-terminal SDE2 is sufficient to suppress the increased PCNA-Ub levels caused by USP1 depletion. HeLa cells were sequentially transfected with USP1 siRNA (vs. control) and HA-C-SDE2 encoding plasmid (vs. empty vector) and analyzed by Western blotting. <b>(F)</b> HeLa cells expressing vector or HA-C-SDE2 were treated with 40 J/m² UVC for 4 h, and PCNA-Ub levels were analyzed by Western blotting. <b>(G)</b> Cells in (F) were plated in 6 well plates, irradiated UVC, and cellular viability was determined by clonogenic survival at 12 days. Data shown are the mean ± SD of three independent experiments. * <i>p</i> < 0.01 compared with empty vector.</p

SDE2 depletion causes replication-associated DNA damage and a defect in replication stress response.

<p><b>(A)</b> SDE2 knockdown leads to increased levels of γH2AX upon DNA damage. HeLa cells transfected with the indicated siRNAs were treated with 40 J/m² UVC for 4 h and analyzed by Western blotting. <b>(B)</b> (left) siRNA-transfected U2OS cells were treated with 40 J/m² UVC for 4 h, and γH2AX foci were analyzed by immunofluorescence. (right) Quantification of cells displaying more than 10 γH2AX foci. Data shown are the mean ± SD from three independent experiments. * <i>p</i> < 0.01 compared with siRNA control. <b>(C)</b> MUS81 depletion suppresses damage-induced γH2AX caused by SDE2 knockdown. HeLa cells transfected with indicated siRNA oligoes were treated with 40 J/m² for 4 h, and cell lysates were analyzed by Western blotting. Note that PCNA monoubiquitination was decreased upon MUS81 knockdown (related to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006465#pgen.1006465.g007" target="_blank">Fig 7</a>). <b>(D, E)</b> Luminescence-based viability (D) or clonogenic survival (E) of siRNA-transfected HeLa cells treated with the indicated doses of DNA damage. Data shown are the mean ± SD from three independent experiments. * <i>p</i> < 0.01 SDE2 knockdown compared with control (except 250 μM HU <i>p</i> < 0.05). <b>(F)</b> SDE2 knockdown causes a defect in S phase progression. HeLa cells transfected with siRNA control or SDE2 were synchronized at G2/M phase by treating 100 ng/mL nocodazole for 16 h. After mitotic shake-off, cells were released into G1 and S phases, and cell cycle was monitored by PI staining and flow cytometry. Data shown are the mean ± SD from three independent experiments. * <i>p</i> < 0.05 for S phase population from cells with SDE2 knockdown vs. control. <b>(G)</b> HeLa cells transfected with siRNA control or SDE2 were left untreated or treated with 40 J/m² UVC, and incubated with 10 μM BrdU for 0.5 h before harvest at 4 h post UVC irradiation. S phase cells were determined by anti-BrdU/PI staining and flow cytometry, and SDE2-depleted BrdU⁺ cells were normalized by control-treated BrdU⁺ cells. Data shown are the mean ± SD from two independent experiments. * <i>p</i> < 0.01 SDE2 knockdown vs. control. <b>(H)</b> Decreased replication recovery of SDE2-depleted cells against UV damage. HeLa cells transfected with siRNA control or SDE2 were pulsed with 10 μM BrdU for 0.5 h, left untreated or treated with 40 J/m² UVC, and released into fresh medium for 4 h. (left) Representative cell cycle distribution measured by anti-BrdU/PI staining and flow cytometry. (right) Relative distribution of early S (A/A+B) and late S (B/A+B) cells out of total BrdU⁺ cells. Data shown are the mean ± SD from three independent experiments. * <i>p</i> < 0.01 for increased early and decreased late S populations from cells with SDE2 knockdown vs. control.</p

SDE2-UBL undergoes CRL4^CDT2-dependent proteolysis.

<p><b>(A)</b> HeLa cells expressing full-length GFP-SDE2 were left untreated or treated with 40 J/m² ultraviolet C (UVC), 2 mM hydroxyurea (HU), or 1 μM mitomycin C (MMC) for the indicated times, and N-terminal GFP-UBL levels were analyzed by Western blotting. <b>(B)</b> HeLa cells exponentially growing or synchronized by nocodazole were collected at the indicated times following mitotic shake-off and analyzed by Western blotting. Cell cycle progression was analyzed by flow cytometry in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006465#pgen.1006465.s004" target="_blank">S3C Fig</a>. <b>(C)</b> (left) Full-length GFP-SDE2 expressing HeLa cells transfected with siRNA CDT2 (vs. control) were treated with 2 mM HU for the indicated times and analyzed by Western blotting. (right) Quantification of immunoblots by Image J. <b>(D)</b> HeLa cells expressing full-length GFP-SDE2 were transfected with siRNA control or CDT2 for 48 h and treated with 50 μg/mL cycloheximide (CHX) for the indicated times. The level of GFP-UBL was analyzed by Western blotting. <b>(E)</b> HeLa cells treated with DMSO or 1 μM MLN4924 were treated with 50 μg/mL CHX for the indicated times, and GFP-UBL levels were analyzed by Western blotting. <b>(F)</b> HeLa cells expressing GFP-SDE2 were transfected with HA-CDT2 plasmid (vs. empty vector), and GFP-UBL levels were analyzed by Western blotting. <b>(G)</b> 293T cells transfected with the indicated plasmids were treated with 10 μM MG132 for 4 h, lysed in a denaturing condition, and incubated with Ni-NTA agarose for the <i>in vivo</i> ubiquitination assay. <b>(H)</b> 293T cells transfected with indicated siRNAs and plasmids were subjected to the <i>in vivo</i> ubiquitination assay.</p

A proposed model for the regulation of SDE2 by PCNA-dependent cleavage and degradation.

<p><b>(A)</b> Targeting of SDE2 to PCNA-associated replication forks via the N-terminal UBL containing a PIP box leads to the cleavage of SDE2 at the diglycine motif. DUB activity is required for its cleavage. <b>(B)</b> The cleaved C-terminal SDE2 functions as a negative regulator of damage-inducible RAD18-dependent PCNA monoubiquitination. The SDE2 domain is required for this process. <b>(C)</b> Degradation of the cleaved N-terminal and C-terminal SDE2 products by CRL4^CDT2 allows timely S phase progression and promotes replication stress response, at least partly through PCNA-Ub-dependent lesion bypass, to ensure genome stability. Deregulation of SDE2 levels, either by knockdown or by defective proteolysis, disrupts this genome maintenance pathway.</p

A PIP box in the SDE2-UBL is required for the cleavage of SDE2.

<p><b>(A)</b> A schematic showing a PIP box in the SDE2-UBL. PIP boxes from different species are aligned, and consensus elements are marked with asterisks. <b>(B)</b> 293T cell lysates expressing GFP-SDE2 GA or GA + PIP (F47A & F48A) mutants were incubated with GST- or GST-PCNA-bound glutathione beads and analyzed by Western blotting. <b>(C)</b> HeLa cells expressing GFP-tagged SDE2 wild-type or PIP mutant were analyzed by Western blotting. <b>(D)</b> GFP-SDE2 variants transfected into U2OS cells were fixed and visualized by fluorescence microscopy. <b>(E)</b> SDE2-Flag proteins <i>in vitro</i> transcribed and translated (IVTT) from reticulocyte lysates were analyzed by Western blotting. Where indicated, ubiquitin vinyl sulfone (Ub-VS) was added during expression. <b>(F)</b> Inhibition of cleavage of IVTT SDE2-Flag GA and PIP mutants. ΔSAP: aa395-450 deletion.</p

Proteasome inhibition blocks expression of Fanconi Anemia/Homologous Recombination genes.

<p>(A) Diagram of NF-kB promoters on <i>FANCD2</i> and <i>BRCA1</i> genes. (B) Expression by qPCR of <i>FANCD2</i> following proteasome inhibition ±30 nM (A549) or 50 nM (NCI-H460) bortezomib or ± inducible <i>PSMA1</i> shRNA, and ±10 Gy IR. All values are normalized to ACTB and all results are mean ± SEM. (C) As in (B) but with BRCA1. (D) NSCLC cell survival following bortezomib and IR is partially rescued by <i>IkBα</i> siRNA knockdown when performed in advance. Cell viability was assayed using an ATP luminescence-based assay measuring relative luminescent units (RLU). All results are mean ± SEM.</p

Knockdown of individual proteasome subunits results in NSCLC cytotoxicity.

<p>Diagram of the 26 S proteasome showing multiple whole genome shRNA screen hits with the following color code: top hit (red), strong hit (>1 shRNA sequence per gene in both cell lines, dark orange), minor hit (1 shRNA sequence per gene in both cell lines, light orange), chymotrypsin-like proteolytic catalytic site (not a hit but highlighted for illustrative purposes, green). Each hit is labeled using the last two alphanumeric characters of the gene’s HUGO nomenclature; e.g., A1 = PSMA1, B5 = PSMB5, M1 = SHFM1.</p