TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

Abstract

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.

Figure 1.

TRAIP interacts with active and stressed replication forks. (A) Analysis of protein recruitment to psoralen–cross-linked chromatin (PSO) compared with an undamaged control (CTR). Chromatin templates were replicated in repair-proficient Xenopus egg extracts. After chromatin reisolation, associated proteins were analyzed by mass spectrometry. Maximal protein intensity is plotted against an overall score determined from several independent experiments. The dotted line indicates the significance threshold (q-value <0.01). (B) Temporal profile of TRAIP intensity on psoralen–cross-linked (red) or undamaged chromatin (blue). (C) Intensity of TRAIP on psoralen–cross-linked chromatin in the presence (black) or absence (red) of the replication inhibitor geminin (GEM). (D) U2OS cells stably expressing GFP-TRAIP were subjected to laser microirradiation, fixed 1 h later, and immunostained with γ-H2AX antibody. (E) As in D, except cells were immunostained with PCNA antibody. (F) As in D, except cells were immunostained with RPA antibody. (G) Representative images of U2OS cells transfected with nontargeting control (CTRL) or TRAIP siRNAs treated or not treated with MMC for 6 h and immunostained with TRAIP antibody. (H) NCC analysis. HeLa S3 suspension cells were incubated with biotin–deoxy-UTP for 15 min and fixed immediately (nascent condition) or cultured for an additional 30 min in the absence or presence of HU before fixation (mature and HU conditions, respectively). Samples were immunoblotted with the indicated antibodies. Bars, 10 µm. MW, molecular weight.

Figure 2.

TRAIP localizes to genotoxic stress sites via a PCNA-binding PIP box. (A) Schematic depiction of human TRAIP showing the PCNA-binding PIP box and its conservation among vertebrates. The residue mutated to generate TRAIP *PIP is highlighted in red. (B) U2OS cells transfected with the indicated combinations of Strep-HA-PCNA and GFP-TRAIP plasmids were subjected to Strep-Tactin pull-down followed by immunoblotting with GFP, HA, and MCM6 antibodies. (C) U2OS cells transfected with GFP-TRAIP constructs containing point mutations in the PIP box (shown in A) were subjected to laser microirradiation, fixed 1 h later, and immunostained with γ-H2AX antibody. Bar, 10 µm. (D) ITC analysis of TRAIP^447–469 fragment binding to PCNA at 25°C. Squares and lines denote the raw measurements and the fitting to a one set of identical sites. The dissociation constant and the thermodynamic parameters of binding are shown. (E) Crystal structure of the PCNA–TRAIP^447–469 complex. Surface representation of the three PCNA protomers and the peptide molecules in sticks colored by atom type are shown. (F) Detailed view of one PCNA protomer and its bound TRAIP^447–469 peptide. Individual residues of the TRAIP^447–469 peptide observed in the crystal structure are indicated. MW, molecular weight.

Figure 3.

TRAIP promotes ssDNA formation and checkpoint signaling after fork stalling. (A) U2OS cells stably expressing RFP-PCNA and GFP-RPA1 were transfected with the indicated siRNAs and exposed to CPT for 1 h before fixation. The intensity of RFP and GFP foci was quantified by ScanR analysis. (B) U2OS cells were transfected with nontargeting control (CTRL) or TRAIP siRNAs, synchronized in S phase by overnight treatment with thymidine, and then released for 4 h before exposure to CPT (cell cycle profiles are shown in Fig. S3 A). 1 h later, cells were fixed and immunostained with RPA (pT21) and PCNA antibodies. (C) U2OS cells transfected with siRNAs, synchronized, and released as in B were subjected to treatment with CPT (1 h), HU (4 h), or MMC (4 h), collected, fractionated into soluble and chromatin-enriched fractions, and immunoblotted with the indicated antibodies. (D) As in B, except BrdU was added to the medium 24 h before fixation. Cells were immunostained with RPA2 and BrdU antibodies under native conditions. (E) Quantification of data in D using QIBC analysis. Results (mean ± SD) from six independent experiments are shown. Bars, 10 µm. MW, molecular weight.

Figure 4.

TRAIP promotes genome stability maintenance after replication stress. (A) U2OS cells transfected with nontargeting control (CTRL) or TRAIP siRNAs were treated with EdU for 20 min and collected. Cells were then processed for EdU detection, stained with DAPI, and analyzed by QIBC. Results (mean ± SD) from three independent experiments are shown. (B) U2OS cells transfected with the indicated siRNAs were labeled with IdU for 10 min, washed extensively, and labeled with CldU for 20 min. DNA fibers were prepared and stained with IdU and CldU antibodies. Fork speed rates were determined by measuring the CldU tract length of IdU-positive fibers. *, P < 0.05; **, P < 0.01. (C) As in B, except that cells were treated with HU for 2 h after washing out IdU and then washed again and incubated with CldU for 1 h. ****, P < 0.0001. (D) U2OS cells transfected with the indicated siRNAs were stained with γ-H2AX antibody and DAPI (left). γ-H2AX–positive micronuclei were quantified by QIBC analysis (right). Results (mean ± SD) from four independent experiments are shown. (E) U2OS cells transfected with the indicated siRNAs were treated or not treated with 60-nM MMC for 24 h and then treated with nocodazole for an additional 2 h and collected. Metaphase spreads were prepared and chromosome aberrations were quantified blindly (top). Red lines indicate the mean of the data plotted. P-values were calculated using a Mann–Whitney U test (n = 50). ***, P < 0.001. A representative metaphase spread from cells treated with TRAIP siRNA and MMC is shown (bottom). B, chromatid break; R, radial chromosome. Bar, 2 µm. (F) Colony formation assay using U2OS cells transfected with the indicated siRNAs and subjected to various doses of MMC for 24 h. Results (mean ± SEM) from three independent experiments are shown.

Figure 5.

Roles of TRAIP ubiquitin ligase activity and PCNA binding in the response to replication stress. (A) Parental U2OS cells (−) and derivative cell lines stably expressing WT or mutant forms of TRAIP resistant to siRNAs targeting endogenous TRAIP (TRAIP^siR) were transfected with nontargeting control or TRAIP siRNAs and analyzed by immunoblotting with antibodies to TRAIP and MCM6 (loading control). (B) Stable U2OS-TRAIP^siR cell lines transfected with TRAIP siRNA were treated with MMC for 6 h, fixed, and coimmunostained with TRAIP and PCNA antibodies. Bar, 10 µm. (C) U2OS cells (−) or derivative U2OS-TRAIP^siR cell lines transfected with nontargeting control or TRAIP siRNAs were synchronized in S phase by overnight treatment with thymidine and then released for 4 h before exposure to CPT. 1 h later, cells were collected, fractionated into soluble and chromatin-enriched fractions, and immunoblotted with the indicated antibodies. (D) As in C, except that cells were treated with HU for 2 h. (E) Colony formation assay using U2OS cells or stable U2OS-TRAIP^siR cell lines transfected with the indicated siRNAs and subjected to various doses of MMC for 24 h. Results (mean ± SEM) from three independent experiments are shown. (F) As in E, except that U2OS-TRAIP^siR WT and ΔRING cell lines were used. (G) As in E, but using U2OS-TRAIP^siR WT and ΔPIP cell lines. (H) U2OS cells or derivative U2OS-TRAIP^siR cell lines transfected with TRAIP siRNA were fixed and stained with DAPI. The proportion of cells with micronuclei was quantified by QIBC analysis. Results (mean ± SD) from three independent experiments are shown. CTRL, control; MW, molecular weight; WCE, whole cell extract.