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. Author manuscript; available in PMC: 2015 Jan 23.
Published in final edited form as: Mol Cell. 2013 Dec 12;53(2):235–246. doi: 10.1016/j.molcel.2013.11.002

PRP19 Transforms into a Sensor of RPA-ssDNA after DNA Damage and Drives ATR Activation via a Ubiquitin-Mediated Circuitry

Alexandre Maréchal 1, Ju-Mei Li 3,#, Xiao Ye Ji 1,#, Ching-Shyi Wu 1, Stephanie A Yazinski 1, Hai Dang Nguyen 1, Shizhou Liu 1,4, Amanda E Jiménez 1, Jianping Jin 3, Lee Zou 1,2,6
PMCID: PMC3946837  NIHMSID: NIHMS545304  PMID: 24332808

Summary

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). While the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 binds RPA directly and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ATR kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, the recovery of stalled replication forks, and the progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR.

Introduction

The stability of the genome relies on the coordinated action of multiple cellular processes, such as DNA replication, DNA repair, and chromosome segregation. When the genome is facing DNA damage or replication stress, concerted cellular responses must be mounted to prevent loss of genomic stability. The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a master regulator of the DNA damage response (DDR) (Cimprich and Cortez, 2008; Flynn and Zou, 2011). ATR, in a complex with its functional partner ATRIP, is activated by a broad spectrum of DNA damage and replication stress. Once activated, ATR phosphorylates Chk1 and other substrates to promote cell cycle arrest, DNA repair, and recovery from replication stress, coordinating the multifaceted DDR. Interestingly, a number of proteins involved in RNA metabolism were recently found to be important for genomic stability or implicated in the DDR by proteomic and genome-wide RNAi screens (Adamson et al., 2012; Beli et al., 2012; Hurov et al., 2010; Matsuoka et al., 2007; Paulsen et al., 2009). These findings raised an intriguing question of whether specific RNA-processing events, or the factors involved in them, directly contribute to the activation or function of the ATR pathway.

RPA-coated single-stranded DNA (RPA-ssDNA), a common intermediate in both DNA repair and the replication stress response, is the key structure that triggers ATR activation (Zou and Elledge, 2003). Impediment of DNA replication forks or compromised activity of replication proteins often leads to exposure of increasing amounts of ssDNA (Byun et al., 2005; Sogo et al., 2002). ssDNA is also generated by nucleolytic processing of stalled or collapsed replication forks, or by processing of DNA nicks and breaks during different types of DNA repair (Costanzo et al., 2003; Giannattasio et al., 2010; Symington and Gautier, 2011). Once exposed in cells, ssDNA is rapidly coated by RPA, presenting a nucleoprotein platform that nucleates the ATRA-TRIP complex and its regulators (Zou and Elledge, 2003). At the junctions of RPA-ssDNA and double-stranded DNA (dsDNA), ATR-ATRIP is juxtaposed to Rad17 and Rad9-Rad1-Hus1 (9-1-1) complexes, which allows TopBP1 to stimulate the kinase activity of ATR and Chk1 phosphorylation (Cotta-Ramusino et al., 2011; Delacroix et al., 2007; Kumagai et al., 2006; Lee et al., 2007; Liu et al., 2011; Mordes and Cortez, 2008). In human cells RPA-ssDNA also interacts with the Mre11-Rad50-Nbs1 (MRN) complex independently of ssDNA/dsDNA junctions, promoting the phosphorylation of RPA32 by ATR (Shiotani et al., 2013). When ATR is activated on RPA-ssDNA, it is perfectly positioned to phosphorylate DNA replication and repair proteins. RPA and several of its binding proteins, such as BLM and SMARCAL1, are ATR substrates and are implicated in the protection of stressed replication forks (Bansbach et al., 2009; Davies et al., 2004; Shiotani et al., 2013; Vassin et al., 2009). Thus, systematic identification of the DDR proteins that associate with RPA-ssDNA will further elucidate how ATR is activated and how it functions during DNA repair and the replication stress response.

In this study, we carried out a proteomic screen for proteins that associate with RPA-ssDNA. The use of both wild-type RPA and a DDR-defective RPA mutant allowed us to identify a large number of RPA-ssDNA-interacting proteins that may participate in the DDR. From this screen, we identified the PRP19 ubiquitin ligase complex as a putative sensor of RPA-ssDNA. PRP19 is an important regulator of pre-mRNA splicing (Chan and Cheng, 2005; Chan et al., 2003; Chen et al., 2006). During splicing, PRP19 ubiquitylates the U4 snRNP component PRP3, leading to stabilization of the U4/U6.U5 snRNP (Song et al., 2010). Furthermore, in both human and yeast cells, PRP19 interacts with RNA polymerase II and couples RNA processing to transcription (Chanarat et al., 2011; David et al., 2011). Interestingly, although PRP19 is a well-characterized regulator of RNA processing, it also has an elusive role in the DDR. In fact, the yeast Prp19 (also known as Pso4) was identified from two independent genetic screens for splicing mutants and DDR mutants (Cheng et al., 1993; Grey et al., 1996). Human PRP19 was also found to be important for genomic stability (Paulsen et al., 2009), and one of its interacting proteins, CDC5L, was shown to affect ATR activation (Zhang et al., 2009). Despite these tantalizing links, whether and how the splicing regulator PRP19 plays a direct role in the DDR remains unknown.

Here, we show that PRP19 binds RPA directly in vitro and localizes to sites of DNA damage via RPA in cells. Independently of its known function in RNA processing, PRP19 promotes ubiquitylation of RPA in a DNA damage-induced manner, and facilitates the accumulation of ATRIP at sites of DNA damage. Depletion of PRP19 compromises the phosphorylation of RPA32 and Chk1, leading to defective recovery of stalled replication forks and impaired fork progression on damaged DNA. Importantly, PRP19 mutants that are unable to bind RPA or function as an E3 ligase failed to support the ATR response, suggesting that the full activation of ATR is driven by a ubiquitylation-mediated circuitry orchestrated by RPA-ssDNA and PRP19. Our results show that an RNA-processing protein in undamaged cells transforms into a DNA damage sensor during the DDR, revealing an unexpected interplay between these two fundamental cellular processes.

Results

A Proteomic Screen for RPA-ssDNA-binding proteins

To better understand how ATR is activated and how it functions on RPA-ssDNA, we sought to systematically identify RPA-ssDNA-binding proteins using a biochemical approach. Biotinylated ssDNA was coated with recombinant human RPA and used to capture binding proteins from HeLa cell nuclear extracts (Fig. 1A). To increase the specificity of this approach, we performed parallel experiments with wild-type RPA (RPAWT) and the t-11 RPA mutant (RPAt-11), which contains 2-amino acid substitutions in the basic cleft of the N-terminal OB-fold of RPA70 (R41E/Y42F) (Haring et al., 2008). RPAt-11 is a separation-of-function mutant that is proficient for DNA replication but defective for the DDR (Haring et al., 2008; Umezu et al., 1998; Xu et al., 2008; Zou and Elledge, 2003). As shown previously, both RPAWT and RPAt-11 bound to ssDNA efficiently (Fig. 1B) (Haring et al., 2008; Zou and Elledge, 2003). The ATR-ATRIP complex, which interacts with the N-terminal OB-fold of RPA70, was only able to bind RPAWT-ssDNA but not RPAt-11-ssDNA (Fig. 1B). In contrast, SMARCAL1, a protein that interacts with the RPA32 subunit of the RPA complex, was efficiently pulled down by both RPAWT-ssDNA and RPAt-11-ssDNA (Fig. 1B) (Bansbach et al., 2009; Ciccia et al., 2009; Yuan et al., 2009; Yusufzai et al., 2009). These results validate the capability of our approach to capture RPA-ssDNA-binding proteins and to identify the proteins that specifically interact with the N-terminal OB-fold of RPA70, a key regulatory module for the DDR.

Fig. 1. A proteomic screen for proteins that associate with RPA-ssDNA.

Fig. 1

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A. Schematic of the screen. Purified heterotrimeric RPA complex was used to coat ssDNA. B. RPAWT- or RPAt-11-coated, biotinylated ssDNA was incubated with HeLa nuclear extracts. The proteins captured by RPA-ssDNA were analyzed using the indicated antibodies. C. The proteins captured by RPAWT-ssDNA, RPAt-11-ssDNA, or beads without ssDNA attached were stained with Coomassie blue. D. Pie chart representation of the top biological functions amongst the RPA-ssDNA-binding proteins generated using the Ingenuity Pathway Analysis Software. Numbers in the pie slices indicate the number of proteins annotated in the specified category. E. A network of DDR and replication proteins was identified by the Ingenuity analysis. Red: proteins exclusively bound to RPAWT-ssDNA; Yellow: proteins preferentially bound to RPAWT-ssDNA; Green: proteins with no preference for RPAWT-ssDNA; See legend of Fig. S1 for additional details on the labeling. Additional information of the proteins identified from the screen is shown in Fig. S1 and Tables S1–S3.

Next, we used mass spectrometry to analyze the proteins captured by RPAWT-ssDNA and RPAt-11-ssDNA (Fig. 1C). From this analysis, we identified 340 proteins bound to RPAWT-ssDNA and 160 proteins bound to RPAt-11-ssDNA (Table S1). In addition to many known RPA-binding proteins (Table S2), we identified a large number of proteins not been previously shown to interact with RPA. We used the Ingenuity Pathway Analysis software (IPA) to divide the RPA-ssDNA-binding proteins into functional groups (Fig. 1D). As expected, these proteins are highly enriched for proteins involved in DNA replication, recombination, and repair (Fig. 1E and Table S1). Interestingly, similar to several recent proteomic and RNAi screens for DDR proteins, our analysis identified a group of proteins involved in RNA processing (Fig. S1) (Adamson et al., 2012; Beli et al., 2012; Hurov et al., 2010; Matsuoka et al., 2007; Paulsen et al., 2009). The results of this proteomic screen provide a useful resource to study the role for RPA-ssDNA in the DDR and other processes.

PRP19 recognizes RPA upon DNA damage

To gain new insights into how RPA-ssDNA regulates ATR, we analyzed the available information on the RPA-ssDNA-binding proteins that we identified. A protein complex containing PRP19 attracted our attention. PRP19 is a U-box-containing E3 ubiquitin ligase that forms a complex with CDC5L, PRL1, and SPF27 (Grote et al., 2010; Vander Kooi et al., 2006). All four components of the PRP19 complex were found to bind RPAWT-ssDNA but not RPAt-11-ssDNA (Fig. 2A). The specific binding of both PRP19 and CDC5L to RPAWT-ssDNA was confirmed by Western blot (Fig. 2A). PRP19 is known to activate pre-mRNA splicing and couples RNA processing to transcription (Chan et al., 2003; Chanarat et al., 2011; David et al., 2011; Song et al., 2010). PRP19 is also important for genomic stability (Paulsen et al., 2009), but whether it has a direct role in the DDR is not known. The binding of the PRP19 complex to RPA-ssDNA raised an interesting question of whether this splicing regulator functions directly in the DDR as a sensor of RPA-ssDNA.

Fig. 2. PRP19 interacts with RPA at sites of DNA damage.

Fig. 2

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A. The PRP19 complex specifically bound to RPAWT-ssDNA but not RPAt-11-ssDNA (left panel). Specific binding of PRP19 and CDC5L to RPAWT-ssDNA was confirmed by Western blot analysis (right panel). B. HEK293T cells transiently expressing SFB-PRP19 were treated with 1 μM CPT for 3 hrs or mock treated. SFB-PRP19 was captured using streptavidin-coated magnetic beads (left panel), and RPA32 was immunoprecipitated (right panel). C. Nuclear extracts were prepared from HeLa cells treated with CPT or mock treated. RPA32 was immunoprecipitated from extracts, and the coprecipitated PRP19 was detected by Western blot. D. HEK293T cells expressing SFB-PRP19 were treated with CPT or mock treated. As indicated, 10 μM of VE-821 was added to cells 30 min before CPT treatment. SFB-PRP19 was captured and the coprecipitated RPA32 was analyzed by Western blot. E. HeLa cells were treated with the transcription inhibitor DRB to reduce the transcription-associated PRP19 signals, and microirradiated with UV laser. Cells were immunostained to visualize protein recruitment to laser tracts. See also Fig. S2.

To test if PRP19 is a sensor of RPA-ssDNA in the DDR, we first assessed the interaction between PRP19 and RPA in cells. Interestingly, while PRP19 is able to bind recombinant RPA-ssDNA in cell extracts, the interaction between SFB (S-Flag-streptavidin binding peptide)-tagged PRP19 and RPA in cells was clearly stimulated by camptothecin (CPT) (Fig. 2B, lanes 4 and 10). In addition to CPT, hydroxyurea (HU) and ultraviolet light (UV) also stimulated the PRP19-RPA interaction in cells (data not shown). Similar to SFB-PRP19, endogenous PRP19 bound to RPA in a CPT-induced manner (Fig. 2C). To understand how the binding of PRP19 to RPA is regulated by DNA damage, we asked if the PRP19-RPA interaction is stimulated by ssDNA or ATR. The interaction between purified PRP19 and RPA was not stimulated by ssDNA in vitro (data not shown). On the other hand, VE-821, a specific inhibitor of ATR (Reaper et al., 2011), clearly diminished the CPT-induced PRP19-RPA interaction in cells (Fig. 2D). These results suggest that the binding of PRP19 to RPA in cells is enhanced by ATR in response to DNA damage.

We next investigated the localization of PRP19 in cells before and after DNA damage. Consistent with its role in splicing, PRP19 was detected in nuclear speckles marked by SC35 in the absence of DNA damage (Fig. S2A) (Spector and Lamond, 2011). PRP3, the substrate of PRP19 in the spliceosome (Song et al., 2010), was colocalized with both PRP19 and SC35 in these nuclear speckles (Fig. S2A). Importantly, upon UV-laser microirradiation, a second population of endogenous PRP19 appeared in punctate foci within γ-H2AX stripes (Fig. 2E). A similar localization pattern was reported for ATR, ATRIP, RPA, and a group of DDR proteins that associate with RPA-ssDNA (Bekker-Jensen et al., 2006). Indeed, endogenous PRP19 colocalized with RPA32 precisely in the microirradiated stripes (Fig. 2E). CDC5L also colocalized with phosphorylated RPA32 in the stripes (Fig. S2B). Although PRP19 loosely associates with the spliceosome, its spliceosome substrate PRP3 did not localize to sites of DNA damage (Fig. S2B). Together, these results show that the PRP19 complex, but not the spliceosome, interacts with RPA at sites of DNA damage.

PRP19 localizes to DNA damage via RPA

PRP19 contains a U-box at the N terminus, a coiled-coil domain in the central region, and seven WD40 repeats at the C terminus. To map the region of PRP19 that is required for RPA binding, we generated a series of SFB-tagged PRP19 fragments and tested their interaction with RPA in CPT-treated cells (Fig. 3A, S3A). The U-box and coiled-coil domain of PRP19 were dispensable for RPA binding. In contrast, either C- or N-terminal truncation of the WD40 domain completely abrogated the interaction with RPA, suggesting that the integrity of this domain is critical for RPA binding. A PRP19 fragment containing only the WD40 domain (WD40WT) was sufficient to bind RPA in CPT-treated cells (Fig. S3B). Furthermore, GST-tagged WD40WT purified from E. coli bound to purified RPA directly (Fig. 3B). Thus, the WD40 domain of PRP19 is both necessary and sufficient for RPA binding.

Fig. 3. PRP19 recognizes DNA damage via its interaction with RPA.

Fig. 3

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A. Schematic of the PRP19 fragments tested and their abilities to bind RPA and CDC5L. B. Purified untagged RPA complex was incubated with GST-WD40WT or GST. The interaction between RPA and GST-WD40WT was tested by GST pulldown. C. Cells expressing full-length SFB-PRP19WT or SFB-PRP19Y405A were treated with CPT, and SFB-tagged proteins were captured from cell extracts. The coprecipitated proteins were analyzed by Western blot. D. HeLa cells were nucleofected with PRP19 siRNA and the indicated plasmids, treated with DRB, and microirradiated with UV laser. See also Fig. S3.

To determine the function of the PRP19-RPA interaction, we sought to disrupt this interaction with mutations in PRP19. Crystal structure of the yeast Prp19 WD40 domain revealed that a conserved region in the fifth blade of the domain is exposed on the protein surface (Vander Kooi et al., 2010). When a tyrosine residue (Y405) of human PRP19 in this conserved region was mutated to alanine in WD40WT, the resulting WD40Y405A mutant displayed reduced affinity to RPA in vitro (Fig. S3C). Furthermore, the full-length PRP19 carrying this point mutation (PRP19Y405A) failed to bind RPA in cells after CPT treatment, but it retained the ability to interact with CDC5L and PRP3 (Fig. 3C). These results suggest that the Y405A mutation specifically disrupts the PRP19-RPA interaction, while preserving the integrity of the PRP19 complex and its ability to interact with the spliceosome. Indeed, PRP19Y405A still localized to nuclear speckles and colocalized with PRP3 in the absence of DNA damage (Fig. S3D). However, in marked contrast to PRP19WT and PRP19ΔUBOX, which lacks the U-box required for E3 ligase activity, PRP19Y405A lost its ability to localize to the laser-microirradiated stripes (Fig. 3D). Together, these experiments demonstrate that PRP19 recognizes DNA damage via its interaction with RPA in cells.

The PRP19 complex promotes RPA and Chk1 phosphorylation

If PRP19 functions as a sensor of RPA-ssDNA, it should be important for certain RPA-ssDNA-directed processes, such as ATR activation. While the interaction between PRP19 and RPA is enhanced by ATR, PRP19 may promote ATR activation by driving a feed-forward loop. To test this possibility, we knocked down PRP19 and CDC5L with siRNAs, and analyzed its effects on the phosphorylation of ATR substrates Chk1 and RPA32. Similar to ATR depletion, knockdown of PRP19 or CDC5L resulted in reduced Chk1 phosphorylation after CPT treatment (Fig. S4A). The CPT-induced RPA32 hyperphosphorylation and the phosphorylation of RPA32 at S4/8, T21, and S33 were all diminished in PRP19 and CDC5L knockdown cells (Fig. 4A). The effects of PRP19 knockdown on RPA32 phosphorylation were confirmed using another siRNA targeting the 3'UTR of PRP19 mRNA, and were suppressed by expression of SFB-PRP19WT (Fig. S4B). In contrast to Chk1 and RPA32, ATM and Chk2 were efficiently phosphorylated in PRP19 and CDC5L knockdown cells after CPT treatment (see Fig. S4E), suggesting that the PRP19 complex regulates activation of ATR but not ATM. Importantly, at the time of analysis, knockdown of PRP19 and CDC5L did not significantly alter the cell cycle and DNA synthesis (Fig. S4C), nor did it affect the splicing of a panel of pre-mRNAs known to undergo exon skipping when the coupling of splicing and transcription is compromised (Fig. S4D) (Dutertre et al., 2010). The levels of a panel of DDR proteins, including those whose levels are sensitive to splicing defects, such as BRCA2 (Adamson et al., 2012), did not change in PRP19 and CDC5L knockdown cells (Fig. S4E). In contrast to PRP19 knockdown, knockdown of PRP3 did not affect Chk1 and RPA32 phosphorylation (Fig. S4F). Collectively, these data suggest that the PRP19 complex may have a direct role in ATR activation independently of its splicing function.

Fig. 4. The PRP19 complex promotes ATR activation and the replication stress response.

Fig. 4

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A. Cells transfected with siRNAs were treated with CPT for 1 hr or mock treated. The levels of various proteins and phosphorylated proteins were analyzed. B–C. Cells transfected with the indicated siRNAs were treated with 2 mM HU for 16 hrs and then released into fresh media. Cells were immunostained for γH2AX foci at 0 and 12 hrs post release (B), and γH2AX-positive cells were quantified (C). For each condition, ≥ 400 cells were counted. The error bars represent standard deviations (SD) from two independent experiments (n=2). D. Cells were labeled with CldU for 30 min in the absence of CPT, and then labeled with IdU for 60 min in the presence or absence of 2.5 μM CPT. DNA fibers were spread onto glass slides, stained, and imaged. The lengths of individual DNA replication tracts were measured. The results of a representative experiment are shown. Numbers in red or green are the median tract lengths for CldU and IdU, respectively. E. The average IdU/CldU length ratios in the indicated cell populations were determined from multiple independent fiber assays. The error bars represent standard deviations (SD) from multiple independent experiments (Control and PRP19: n=3; CDC5L: n=2). See also Fig. S4.

In response to CPT, resection of DNA double-stranded breaks (DSBs) is a prerequisite for RPA phosphorylation. Resection and the post-resection phosphorylation of RPA can be monitored using RPA32 foci and phospho-RPA32 foci, respectively. Knockdown of CtIP, a protein required for resection (Sartori et al., 2007), did not alter the induction of DSBs by CPT, but reduced both RPA32 foci and phospho-RPA32 foci (Fig. S4G–L). In contrast, PRP19 and CDC5L knockdown cells displayed only a mild reduction in RPA32 foci, but a dramatic reduction in phospho-RPA32 foci (Fig. S4G–L). Importantly, in most of the PRP19 or CDC5L knockdown cells that displayed CPT-induced RPA32 foci, phospho-RPA32 foci were not detected (Fig. S4M–N). Together, these results suggest that the PRP19 complex primarily functions in the post-resection phase of the DDR.

The PRP19 complex facilitates DNA replication under stress

Our finding that PRP19 is an important regulator of the ATR response prompted us to investigate if PRP19 regulates DNA replication under stress. We analyzed the ability of PRP19 and CDC5L knockdown cells to recover from HU-induced replication arrest. Both PRP19 and CDC5L knockdown cells progressed through S phase more slowly than control cells after release from HU (Fig. S4O). Furthermore, at 12 hours after the release from HU, γH2AX foci largely disappeared from control cells, but persisted in PRP19 and CDC5L knockdown cells (Fig. 4B–C). These results suggest that the PRP19 complex is important for the recovery from replication arrest.

We next used the DNA fiber assay to monitor progression of replication forks in PRP19 and CDC5L knockdown cells. Newly synthesized DNA was first labeled with CldU in cells for 30 minutes prior to CPT treatment, and then labeled with IdU for 1 hour in the absence or presence of CPT (Fig. 4D). In the absence of CPT, neither PRP19 nor CDC5L knockdown significantly altered replication fork progression (Fig. 4E, S4P). In the presence of CPT, however, forks progressed significantly more slowly in PRP19 and CDC5L knockdown cells than in control cells (Fig. 4D–E). In addition, after CPT treatment, the replication tracts indicative of stalled or collapsed forks (0–5 μm IdU tracts) were significantly increased in PRP19 and CDC5L knockdown cells compared with control cells (Fig. 4D, S4Q). Together, these results demonstrate that the PRP19 complex is critical for fork progression on damaged DNA.

PRP19 functions as an RPA-ssDNA-sensing ubiquitin ligase in ATR activation

The ability of PRP19 to recognize DNA damage via RPA and its role in ATR activation and the replication stress response prompted us to ask if PRP19 functions as a sensor of RPA-ssDNA in these processes. In cells depleted of endogenous PRP19, expression of siRNA-resistant SFB-PRP19WT efficiently restored CPT-induced RPA32 and Chk1 phosphorylation (Fig. 5A, S5A). In contrast to PRP19WT, the PRP19Y405A mutant that is unable to bind RPA failed to restore RPA32 and Chk1 phosphorylation (Fig. 5A, S5A). Moreover, the PRP19ΔUBOX mutant, which is inactive as an E3 ligase but retains the ability to interact with RPA and localize to sites of DNA damage, also failed to restore RPA32 and Chk1 phosphorylation (Fig. 5B, S5B). At 12 hours after the release from HU arrest, γH2AX and RAD51 foci largely disappeared from control cells, but persisted in a significant fraction of PRP19 knockdown cells (Fig. 5C–D). In contrast to PRP19WT, neither PRP19Y405A nor PRP19ΔUBOX suppressed the defect in resolving γH2AX and RAD51 foci (Fig. 5C–D), demonstrating that PRP19 indeed functions as an RPA-ssDNA-sensing ubiquitin ligase to promote ATR activation and recovery of stalled or collapsed replication forks.

Fig. 5. PRP19 promotes ATR activation and the replication stress response as an RNA-ssDNA-sensing ubiquitin ligase.

Fig. 5

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A–B. Cells were nucleofected with control or PRP19 siRNA and plasmids expressing siRNA-resistant SFB-PRP19WT, SFB-PRP19Y405A (A), or SFB-PRP19ΔUBOX (B). Cells were subsequently treated with 1 μM CPT for 2 hrs, and analyzed by Western blot using the indicated antibodies. C–D. Cells were nucleofected as in (A) and then treated with 2 mM HU for 16 hrs. Cells were then released into fresh media and immunostained for γH2AX and RAD51 foci at 0 and 12 hrs. For each condition, ≥ 300 cells were counted. The error bars represent standard deviations (SD) from two independent experiments (n=2). See also Fig. S5.

PRP19 ubiquitylates RPA and promotes ATRIP recruitment

The requirement of PRP19 ubiquitin ligase activity for ATR activation raised the question as to what is ubiquitylated by PRP19. RPA32, RPA70, and ATRIP were shown to be ubiquitylated by proteomic studies (Povlsen et al., 2012; Vasilescu et al., 2007). Overexpression of PRP19 enhanced ubiquitylation of RPA32 and RPA70, but not ATRIP (Fig. 6A, lanes 5 and 6; S6A–B). After CPT treatment, the ubiquitylation of RPA32 was stimulated in cells overexpressing PRP19, suggesting that PRP19 promotes RPA32 ubiquitylation in a DNA damage-induced manner (Fig. 6A, lanes 4 and 6; see Fig. 6C, lanes 2 and 3). Importantly, the ubiquitylation of myc-tagged and endogenous RPA32 in CPT-treated cells was reduced by PRP19 knockdown (Fig. 6B–C), suggesting that PRP19 is required for the DNA damage-induced RPA32 ubiquitylation. PRP19 is known to function with the E2 ubiquitin-conjugating enzyme UbcH5c and modify PRP3 with K63-linked ubiquitin chains (Song et al., 2010). We found that wild-type (WT) ubiquitin, but not the K63R ubiquitin mutant, was efficiently conjugated to RPA32 in a DNA damage-induced manner (Fig. 6D), showing that RPA32 is ubiquitylated with K63-linked chains during the DDR. In addition, purified PRP19 complex and UbcH5c were able to ubiquitylate RPA32 and RPA70 in vitro (Fig. 6E, S6C). It should be noted that RPA32 was primarily mono-ubiquitylated by PRP19 and UbcH5c in vitro (Fig. 6E), suggesting that additional regulations and/or factors are needed for the efficient poly-ubiquitylation of RPA32 in vivo. Nonetheless, it is clear that RPA32 is ubiquitylated with K63-linked chains during the DDR in a PRP19-dependent manner.

Fig. 6. PRP19 is required for DNA damage-induced RPA ubiquitylation.

Fig. 6

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A. Cells were transfected with the indicated plasmids, treated with 1 μM CPT for 3 hrs or mock treated, and then processed for anti-HA immunoprecipitation under denaturing conditions. B. Cells were first transfected with the indicated plasmids and subsequently retransfected with control or PRP19 siRNA, treated with CPT and processed for anti-HA immunoprecipitation under denaturing conditions. C–D. Cells were transfected with the indicated plasmids and subsequently retransfected with control or PRP19 siRNA. Cells were then treated with CPT and processed for Ni-NTA pulldown under denaturing conditions. E. The PRP19 complex was affinity purified from HEK293T cells expressing SFB-PRP19. The ubiquitylation reactions were performed by incubating the PRP19 complex with in vitro translated HA-RPA32, ubiquitin, ubiquitin aldehyde, ATP and energy regeneration system at 30°C for 30 min. UbcH5c or the Ubc13-Mms2 complex were used as E2. See also Fig. S6.

To pinpoint how PRP19-mediated ubiquitylation promotes ATR activation, we asked if PRP19 and CDC5L are required for ATR autophosphorylation, an early event of ATR activation that depends on the recruitment of ATR-ATRIP to RPA-ssDNA (Liu et al., 2011; Nam et al., 2011). Knockdown of PRP19 or CDC5L reduced ATR phosphorylation at T1989 (Fig. 7A), suggesting that the recruitment of ATR-ATRIP to RPA-ssDNA is compromised. Consistently, CPT-induced focus formation of GFP-ATRIP was reduced in PRP19 and CDC5L knockdown cells (Fig. 7B–C). The defect in ATRIP focus formation was suppressed by PRP19WT, but not PRP19Y405A and PRP19ΔUBOX, suggesting that PRP19 acts as an RPA-ssDNA-sensing ubiquitin ligase to promote the accumulation of ATR-ATRIP at sites of DNA damage (Fig. 7D, S7A). Because PRP19 promotes K63-linked ubiquitylation of RPA32, we asked if ATR-ATRIP has an affinity for this type of ubiquitin chain. Biotinylated K48- or K63-linked ubiquitin chains were used as bait to pull down proteins from nuclear extracts. Only the K63-linked chain, but not the K48-linked chain, was able to capture ATRIP from nuclear extracts (Fig. 7E). Furthermore, purified Flag-ATRIP bound directly to the K63-linked chain, but not the K48-linked chain (Fig. S7B). Thus, the K63-linked ubiquitin chains of RPA32 may enhance the recruitment and/or retention of ATRIP as a DNA damage-induced anchor, explaining how PRP19 promotes ATR activation as a ubiquitin ligase on RPA-ssDNA.

Fig. 7. The PRP19 complex promotes the recruitment of ATR-ATRIP to sites of DNA damage.

Fig. 7

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A. HCT116 cells transfected with control, PRP19, or CDC5L siRNA were treated with CPT for 2 hrs or mock treated. Endogenous ATR was immunoprecipitated from cell extracts and analyzed with ATR and ATR pT1989 antibodies. B. HeLa cells were transfected with the indicated siRNAs and a plasmid expressing GFP-ATRIP. Cells were treated with 1 μM CPT for 1 hr and analyzed for GFP signals. C. GFP-positive cells with GFP-ATRIP foci were quantified and shown. For each condition, ≥ 400 cells were counted. The error bars represent standard deviations (SD) from two or three independent experiments (Control and PRP19: n=3; CDC5L: n=2). D. HeLa cells were transfected with the indicated siRNAs, and 24 hrs later were retransfected with a GFP-ATRIP-expressing plasmid together with the indicated plasmids. The levels of the relevant proteins are shown in Fig. S7A. Transfected cells were treated with CPT and analyzed for GFP signals. For each condition, ≥ 250 cells were counted. The error bars represent standard deviations (SD) from three independent experiments. An unpaired two-tailed t-test was used to evaluate significance *: P ≤ 0.05, **: P ≤ 0.01. E. Biotinylated tetra-ubiquitin chains were incubated with nuclear extracts from HeLa cells. Proteins bound to ubiquitin chains were pulled down using streptavidin-conjugated magnetic beads and analyzed by Western Blot. F. A model for the ATR- and PRP19-mediated feed-forward loop that promotes ATR-ATRIP recruitment and activation. See also Fig. S7.

Discussion

PRP19 links ubiquitylation to the RPA-ssDNA platform

The localization of DDR proteins to sites of DNA damage plays a key role in the spatial and temporal regulations of the DDR. The two best-characterized platforms for the DDR are the γH2AX-decorated chromatin and RPA-ssDNA (Ciccia and Elledge, 2010). While RPA-ssDNA is known to interact with ATR-ATRIP and a number of DDR proteins involved in DNA damage signaling and DNA repair, a comprehensive understanding of the RPA-ssDNA-interacting proteins is still lacking. It is important to note that the RPA-ssDNA complex is a nucleoprotein structure induced by DNA damage and replication stress, and that the binding properties of RPA-ssDNA are likely distinct from those of free RPA. In this study, we carried out a unique proteomic screen using the RPA-ssDNA complex as bait. Furthermore, the use of a DDR-defective RPA point mutant allowed us to identify a large number of RPA-ssDNA-interacting proteins with high confidence. This screen not only identifies the PRP19 complex as a sensor of DNA damage, but also provides a rich resource for future studies to further elucidate the functions of the RPA-ssDNA platform in the DDR and other cellular processes.

The finding of the PRP19 ubiquitin ligase as a sensor of RPA-ssDNA brings us an extended view of how RPA-ssDNA functions in the DDR. The function of the γH2AX platform relies on a ubiquitylation cascade mediated by the ubiquitin ligases RNF8 and RNF168 (Lukas et al., 2011). Our finding that PRP19 functions as an RPA-ssDNA-sensing ubiquitin ligase to promote ATR activation suggests that RPA-ssDNA is also a platform for ubiquitylation. Furthermore, our results reveal that the ubiquitylation events on γH2AX and RPA-ssDNA share remarkable similarities. In response to DSBs, RNF8 and RNF168 are recruited by γH2AX to the chromatin flanking DSBs, driving the formation of K63-linked ubiquitin chains on histones H2A and H2AX (Doil et al., 2009; Huen et al., 2007; Mailand et al., 2007; Stewart et al., 2009). Similarly, the PRP19 complex recognizes RPA-ssDNA in a DNA damage-induced and ATR-stimulated manner, promoting the formation of K63-linked ubiquitin chains on RPA32 (Fig. 7D). The strong parallels between the ubiquitylation circuitries on γH2AX and RPA-ssDNA suggest that both of these DDR platforms serve to recruit ubiquitin ligases and provide substrates for ubiquitylation, revealing that the DDR is driven by a common mechanism shared by the two key platforms.

Dual recognition of ubiquitylated RPA-ssDNA by ATRIP

ATRIP interacts with RPA directly and plays a key role in the recruitment of ATR-ATRIP to sites of DNA damage (Zou and Elledge, 2003). However, the localization of ATRIP to sites of DNA damage is compromised in cells depleted of PRP19 and cells in which PRP19 fails to recognize RPA or function as a ubiquitin ligase, suggesting that a ubiquitin-mediated circuitry on RPA-ssDNA is also needed for efficient ATR-ATRIP recruitment in vivo. The interaction between PRP19 and RPA is enhanced by ATR, suggesting that PRP19 is part of an ATR-driven feed-forward loop that promotes full activation of ATR. Importantly, PRP19 is required for the formation of K63-linked ubiquitin chains on RPA32 in response to DNA damage, and ATRIP has an affinity for this type of ubiquitin chain. These results suggest that the K63-linked ubiquitin chains on RPA and perhaps other PRP19 substrates may serve as a DNA damage-induced anchor for ATR-ATRIP to enhance its recruitment and/or retention (Fig. 7F). Together, our findings suggest a revised model for the recruitment of ATR-ATRIP to RPA-ssDNA. In this model, ATR-ATRIP not only binds RPA-ssDNA directly, but also recognizes RPA-ssDNA through ubiquitin chains. ATR-ATRIP and PRP19 constitute a mutually reinforcing feed-forward loop on RPA-ssDNA to enhance the recruitment/retention of each other and to drive the full activation of ATR (Fig. 7F).

The abilities of ATRIP to bind RPA directly and recognize the specific type of ubiquitin chain on RPA suggest a `dual-recognition' mechanism for ATRIP recruitment. This mechanism is remarkably similar to the mechanisms by which many other DDR proteins are recruited. Ubiquitylated PCNA plays an important role in the recruitment of the DDR proteins that are involved in post-replicative repair. Many of the DDR proteins recruited by ubiquitylated PCNA, such as Pol η, Pol ι, Pol κ, Spartan, and ZRANB3, contain at least one motif that binds PCNA and another motif that recognizes ubiquitin (Bienko et al., 2005; Centore et al., 2012; Ciccia et al., 2012; Ghosal et al., 2012; Juhasz et al., 2012; Machida et al., 2012; Plosky et al., 2006). Similarly, a number of the DDR proteins that are recruited to the ubiquitylated chromatin flanking DSBs, such as RNF168, RAD18, and RAP80, use tandem protein interaction modules to engage ubiquitin chains or ubiquitylated nucleosomes (Panier et al., 2012). Furthermore, it was recently shown that 53BP1 uses two distinct motifs to recognize ubiquitylated histone H2A and methylated histone H4 (Fradet-Turcotte et al., 2013). All these examples argue that the dual-recognition mechanism is critical to provide the specificity and affinity that are necessary for the efficient recruitment of DDR proteins. Based on our findings on ATRIP and PRP19, we suggest that ubiquitylated RPA-ssDNA is recognized by ATRIP and perhaps other DDR proteins through the dual-recognition mechanism, potentiating the function of this DDR platform.

Is PRP19 a link between the DDR and RNA processing?

PRP19 regulates transcription-coupled RNA processing in the absence of DNA damage (David et al., 2011), but it is quickly transformed into a sensor of RPA-ssDNA when DNA damage emerges. This DNA damage-induced transformation of PRP19 raises an interesting possibility that the DDR, transcription, and RNA processing are functionally linked via PRP19. In yeast, both Prp19 and RPA preferentially associate with transcribed genes in the genome in the absence of DNA damage (Chanarat et al., 2011; Sikorski et al., 2011). In human cells, RPA was shown to be preferentially localized to transcribed genes in response to DNA damage (Jiang and Sancar, 2006). These observations imply that RPA-ssDNA is transiently generated during transcription or when transcription encounters DNA damage, DNA replication forks, or other impediments. One example of such a scenario is the formation of R-loops, which is induced by stable RNA:DNA hybrids during transcription (Aguilera and Garcia-Muse, 2012). The unique abilities of PRP19 to associate with the transcription machinery and to recognize RPA-ssDNA may poise it as a key sensor of genomic instability during transcription. Furthermore, if the recruitment of PRP19 to sites of DNA damage moves it away from the transcription machinery, this may provide a mechanism to repress transcription, RNA processing, or both of these processes during the DDR. Further studies on the PRP19 complex will allow us to explore these exciting possibilities.

Experimental Procedures

RPA-ssDNA pulldown

RPA-ssDNA pulldown was performed as previously described (Yang and Zou, 2006). The LC-MS/MS analysis was performed at the Taplin Biological Mass Spectrometry facility of Harvard Medical School.

Ubiquitylation analysis

Cells were lysed by boiling in lysis buffer (2% SDS, 150 mM NaCl, 10 mM Tris HCl pH 8.0) or by incubating in guanidium-HCl lysis buffer (6 M Guanidium HCl, 20 mM Tris HCl pH 8.0, 0.5 M NaCl, 5% Glycerol, 25 mM Imidazole) for experiments using HA- or His6-tagged ubiquitin respectively. Following sonication, precipitation of HA-ubiquitylated proteins was carried out in lysis buffer 1:10 in dilution buffer (10 mM Tris HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) using anti-HA probe conjugated agarose beads (Santa Cruz Biotechnology). Beads were washed in washing buffer (10 mM Tris HCl pH 8.0, 1 M NaCl, 1 mM EDTA, 1% NP-40) and resuspended in Laemlli buffer. Following sonication, Ni-NTA denaturing pulldown was carried out directly in guanidium-HCl buffer using Ni-NTA agarose resin (Invitrogen). Precipitates were washed twice in guanidium-HCl buffer containing 0.1% Tween-20, twice in buffer B (guanidium HCl buffer 1:4 in buffer C) and twice in buffer C (25 mM Tris-HCl pH 6.8, 150 mM NaCl, 25 mM Imidazole, 10 mM NEM, 5% Glycerol, 0.1% Tween-20) before resuspension in Laemlli buffer.

UV-laser microirradiation

Cells were incubated in DMEM containing 10 μM BrdU 24 hrs prior to the microirradiation. Before microirradiation, media was replaced with DMEM without phenol red. Microirradiation was performed using a 355-nM UV laser on an Arcturus Veritas Laser Capture Microscope (Life technologies) at the Advanced Tissue Resource Center of the Harvard NeuroDiscovery Center.

Supplementary Material

01
02

Highlights

  1. PRP19 is identified as a sensor of RPA-ssDNA from a proteomic screen.

  2. PRP19 recognizes DNA damage via its interaction with RPA.

  3. PRP19 regulates ATR activation as a ubiquitin ligase.

  4. PRP19 promotes RPA ubiquitylation and ATRIP recruitment after DNA damage.

Acknowledgements

We thank Drs. S. Elledge, J. Chen, M. Wold, X. Wu, and W. Wang for reagents, and members of the Zou lab for helpful discussions. AM is supported by a postdoctoral fellowship from the FRSQ, Québec, Canada. SAY is a recipient of a postdoctoral fellowship from the Department of Defense (BC120504). JJ is a Pew Scholar, and LZ is a Jim & Ann Orr Massachusetts General Hospital Research Scholar. This work was supported by grants from the Welch Foundation (AU-1711) and NIH (GM102529) to JJ, and grants from NIH (GM076388) and the Federal Share of MGH Proton Program to LZ.

Footnotes

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Additional experimental procedures are provided in the supplementary information.

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Supplementary Materials

01
NIHMS545304-supplement-01.pdf (12.4MB, pdf)
02
NIHMS545304-supplement-02.xlsx (22.8KB, xlsx)

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