Regulation of PCNA polyubiquitination in human cells
© Gray et al; licensee BioMed Central Ltd. 2010
Received: 27 March 2009
Accepted: 30 March 2010
Published: 30 March 2010
The ubiquitin-based molecular switch dictating error free versus error prone repair has been conserved throughout eukaryotic evolution. A central component of this switch is the homotrimeric clamp PCNA, which is ubiquitinated in response to genotoxic stress allowing recovery of replication forks blocked at sites of DNA damage. The particulars of PCNA ubiquitination have been elucidated in yeast and to a further extent recently in human cells. However, gaps in the detailed mechanism and regulation of PCNA polyubiquitination still persist in human cells.
We expand upon several studies and show that PCNA is polyubiquitnated in normal skin fibroblasts, and that this ubiquitination is dependant on RAD18. Furthermore we define the types of DNA damage that induce ubiquitination on PCNA. Cisplatin, methylmethane sulphonate and benzo(a)pyrene-diol-epoxide induce the polyubiquitination of PCNA to the same extent as UV while polyubiquitination is not detected after X-ray treatment. Moreover, we show that ubiquitination of PCNA is not regulated by cell cycle checkpoint kinases ATM-Chk2 or ATR-Chk1. Significantly, we report that PCNA polyubiquitination is negatively regulated by USP1.
Our results demonstrate the importance of PCNA polyubiquitination in human cells and define the key regulator of this ubiquitination.
In recent studies PCNA ubiquitination has been identified as an important modification in human cells [1–4]. Similar to yeast, human PCNA is monoubiquitinated by hRad6/hRad18 (governs error-prone repair) which increases the affinity for translesion polymerase Polη and enables a polymerase switch in response to DNA damage . Moreover hRad18 also interacts with Polη thereby facilitating its localization to sites of DNA damage . PCNA is also polyubiquitinated by the hMms2/hUbc13/SHPRH or hMms2/hUbc13/HLTF complex, which govern error-free repair and disruption of this polyubiquitination leads to genomic instability resulting in increased mutagenesis and gross chromosomal rearrangements [1, 2, 6, 7]. We have previously used cancer cell lines to demonstrate PCNA polyubiquitination. However, the use of cancer cells may result in a distortion of the physiological damage response. Therefore, it remains to be determined whether PCNA polyubiquitination is important in normal human fibroblasts and if this potential modification depends on RAD18.
Another central question is how human cells regulate mono and polyubiquitination of PCNA and hence how they induce or limit the deployment of DNA repair machinery in the presence or absence of damage. One possibility is that PCNA ubiquitination is regulated by cell cycle checkpoint kinases ATR-Chk1 or ATM-Chk2 given their central role in damage surveillance [8–11]. An alternative possibility is deubiquitinating enzymes (DUBs). DUBs are cysteine proteases that cleave ubiquitin from mono and polyubiquitinated substrates. In 2006 Huang et al., were the first to reveal that USP1 negatively regulates monoubiquitination of PCNA in the absence of DNA damage in order to control TLS .
In this study we examined the details of PCNA polyubiquitination in normal human fibroblasts and cancer cell lines. Contrary to previous studies, we found that PCNA is mono and polyubiquitinated in primary human skin fibroblasts after UV irradiation and that this modification is dependant on RAD18. In addition, we found that PCNA is polyubiquitinated in response to a variety of DNA damaging agents and that polyubiquitination occurs on K164. Since cell cycle checkpoint kinases are predominantly activated after DNA damage we also sought to determine whether PCNA ubiquitination is regulated by global sensors such as ATR or ATM. Similar to studies in Xenopus, S. pombe and human cells, we report that mono or polyubiquitination of PCNA is not regulated by the cell cycle checkpoint kinases in human cells [13–15]. Significantly, we confirm that the candidate DUB for negatively regulating PCNA polyubiquitination is USP1.
Cell Culture, treatments and transfections
The A549, HEK 293T and HeLa, HCT116, GM038 (skin fibroblasts passage 15) cell lines were cultured in DMEM (Gibco, Invitrogen, Carlsbad, California, United States) supplemented with 10% FBS (Gibco, Invitrogen, Carlsbad, California, United States) and 1× Pen/Strep (Gibco, Invitrogen, Carlsbad, California, United States). UV irradiation (30 J/m2) was performed using a UVC germicidal lamp at a fluence rate of 1 J/m2/s. Cisplatin (obtained from the Ottawa Hospital Pharmacy, Ottawa, Ontario, Canada) and mitomycin C (Sigma, St. Louis Missouri, United States) were added to cells for 3 hours at a dose of 160 μM and 0.04 μg/ml, respectively, after which they were washed with PBS and supplemented with fresh media. Cisplatin and MMC treated cells were lysed 3 hours post treatment. MMS (Sigma, St. Louis Missouri, United States) was added directly to cells up to 0.02% for 45 minutes followed by immediate lysis. Cells were irradiated with either 4 or 10 Gray of ionizing radiation and harvested 6 hours post irradiation. For caffeine treatment, cells were incubated for 1 hour with either 2.5, 10 or 20 mM caffeine (Sigma, St. Louis Missouri, United States) prior to UV irradiation (30 J/m2). GM038, 293T and Hela cells were transfected with siGENOME SMARTpool reagent specific for either human RAD18 or USP1 (Dharmacon Research, Lafayette, Colorodo, United States) using oligofectamine (Invitrogen, Carlsbad, California, United States). The transfections were performed 72 hours prior to harvesting the cells to achieve optimal long-term knockdown. 293T were also transiently transfected with either a GFP-tagged WT PCNA or K164R PCNA DNA plasmid using GeneJuice transfection reagent (Novagen) as per company protocol.
UV irradiation induces PCNA polyubiquitination in normal fibroblasts
PCNA polyubiquitination is dependant on RAD18 in primary fibroblasts
In our previous study we demonstrated that disruption of RAD18 abrogates PCNA polyubiquitination in several cancer cell lines after UV irradiation . Therefore, we sought to determine whether this was the case in human fibroblasts. GM38 cells were targeted with siRNA against RAD18. As expected, RAD18 knockdown resulted in a substantial decrease in the mono and di-ubiquitinated species (Figure 1C). Therefore, PCNA polyubiquitination seems to be an important physiological response to UV damage in healthy cells.
PCNA is polyubiquitinated in response to a variety DNA damaging agents
PCNA polyubiquitination is regulated by USP1 and not by cell cycle checkpoint kinases
In a previous study we were the first to show that K63-linked polyubiquitination is important in skin fibroblasts but did not demonstrate the substrate involved in these cells . Here we expand upon that study and demonstrate that PCNA is the substrate for K63-linked polyubiquitination and that PCNA polyubiquitination is a normal physiological response to DNA damage in normal diploid skin fibroblast and that this process is dependant on RAD18. Moreover, we report that PCNA is polyubiquitinated in response to a variety of genotoxic agents that distort DNA including UV, CPT, MMS and BPDE. This would suggest that PCNA polyubiquitination is a common mechanism used to protect cells against the mutagenic effects of DNA damage.
Thus far our data have shown that PCNA polyubiquitination is induced by several DNA damaging agents in a variety of human cell lines. Interestingly, the same replication stressors used in this study also activate a global checkpoint response which is governed by two serine/threonine kinases ATM and ATR. Their activation facilitates the recruitment and phosphorylation of other proteins, including Chk1 and p53, which consequently arrest cells at G1 [20, 21]. Our interest in this pathway in regulating PCNA ubiquitination stems from studies which demonstrated that ATM and ATR activation are necessary for phosphorylation and subsequent ubiquitination of FancD2 . Since FancD2 colocalizes with PCNA after DNA damage we postulated that ATR or ATM may similarly regulate PCNA ubiquitination. Inhibition of ATR or ATM with caffeine showed no disruption in PCNA ubiquitination. In fact it showed a marginal increase in both PCNA mono and polyubiquitination, at least in A549 cells. These results were similar to those recently reported by Chang et al., and Niimi et al [14, 15]. However, this does not rule out that PCNA ubiquitination is regulated by cell cycle checkpoint kinases; rather, it may point to alternative pathways that may compensate for the inhibition of ATM and ATR. Recently, the p38 SAPK-MK2 pathway was demonstrated to be involved in the response to DNA damage converging on similar substrates activated by ATR and ATM . Since caffeine has been shown to upregulate p38 activity one could postulate that the p38 pathway is compensating for the loss of ATR/ATM . Future studies will be required to determine whether inhibition of ATR, ATM and the p38 pathway in combination abrogates PCNA ubiquitination.
Recent studies point towards DUBs as another means to regulate PCNA ubiquitination . For example, USP1 has been implicated in the constitutive deubiquitination of monoubiquitinated PCNA and USP1 functions independently of the cell cycle checkpoint kinases similar to PCNA polyubiquitination [12, 15]. Here we show that USP1 is also involved in deubiquitinating polyubiquitinated PCNA, the implications of which are unknown. Functionally, Huang et al. showed that disruption of USP1 lead to increased spontaneous and UV induced mutagenesis . They postulated that the increase in mutagenesis could be due to the dysregulated function of Polη or recruitment of other error-prone polymerases to the damaged sites . In light of our data showing an increase in PCNA polyubiquitination after USP1 disruption, another explanation for the increased mutagenesis could be the contribution of unscheduled, illegitimate and dysregulated homologous recombination. During the writing of this manuscript Motegi et al, also published corroborating data demonstrating an increase in polyubiquitination of PCNA after abrogating USP1 expression .
In conclusion, our data show that PCNA polyubiquitination is an important response to DNA damage and that it is constitutively regulated by USP1 and not by cell cycle checkpoint kinases. A challenge for future investigations will be to elucidate whether PCNA ubiquitination is a compartmentalized response simply regulated by USP1 alone or regulated by other signaling pathways.
proliferating cell nuclear antigen
ubiquitin specific protease
benzo(a) pyrene diol-epoxide
DNA damage tolerance
The GFP-PCNA and GFP-K164R PCNA constructs were kindly provided by Dr. Roland Kaanars (University of Rotterdam). This work was funded by the National Cancer Institute of Canada (grant number 014132).
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