MOLECULAR & CELLULAR ONCOLOGY 2016, VOL. 3, NO. 1, e1063571 (2 pages) http://dx.doi.org/10.1080/23723556.2015.1063571
AUTHOR’S VIEW
Damaged replication forks tolerate USP7 to maintain genome stability Anastasia Zlatanou and Grant S. Stewart School of Cancer Sciences, University of Birmingham, Birmingham, UK
ABSTRACT
ARTICLE HISTORY
RAD18 functions to promote DNA damage tolerance (DTT), a process that ensures faithful genome duplication. Protein ubiquitylation/deubiquitylation is a critical regulatory mechanism controlling DTT. Recently, we have identified the deubiquitylating enzyme USP7 as a component of the DTT machinery that acts to protect RAD18 from proteasome-dependent degradation.
Received 11 June 2015 Revised 15 June 2015 Accepted 15 June 2015
Obstacles that interfere with DNA replication can have detrimental consequences for genomic stability and cell viability. Replication fork progression can be hindered by the presence of bulky lesions because the replicative polymerases are unable to synthesize across a damaged DNA template. Cells employ a variety of mechanisms to tolerate and bypass these lesions, collectively designated DNA damage tolerance (DTT), in order to allow the completion of DNA synthesis. These include the recruitment of alternative polymerases that can synthesize across the lesion or a temporary switch of DNA synthesis to use the undamaged nascent DNA as a template.1 The E3 ubiquitin ligase RAD18 regulates both branches of DDT, primarily through its ability to monoubiquitylate proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity factor. This modification of PCNA facilitates the temporary replacement of replicative polymerases with trans-lesion synthesis (TLS) polymerases, which are able to replicate across the lesion albeit at the cost of increased mutagenesis due to their poor fidelity and lack of an exonuclease proofreading activity. Further ubiquitylation of PCNA by two other ubiquitin ligases that associate with RAD18— SHPRH and HLTF—promotes a less-characterized mode of lesion bypass that is error-free and is termed ‘template switching’. This pathway involves the replicative polymerase and uses the newly synthesized daughter strand as an undamaged DNA template in order to synthesize past the lesion. Polyubiquitylation, particularly with K48-linked ubiquitin chains, is primarily associated with targeting proteins for proteasome-dependent degradation. However, little is known about how it contributes to the control of DNA replication in the presence of potentially deleterious genetic damage. We have recently reported that RAD18 undergoes polyubiquitylation and proteasomal degradation unless it is protected by the catalytic activity of the deubiquitylating enzyme ubiquitin-specific peptidase 7 (USP7, also known as HAUSP).2 USP7 has been implicated in the regulation of multiple different cellular pathways including viral integration, DNA repair, apoptosis, and epigenetic transmission. This function is primarily mediated through its ability to regulate the stability of its interacting partners by dismantling polyubiquitin chains attached to them.3 Consistent with this, our work demonstrates that a
CONTACT Grant S. Stewart © 2016 Taylor & Francis Group, LLC
[email protected]
significant fraction of RAD18 becomes unstable in the absence of USP7 and, as a result, USP7-depleted cells phenocopy RAD18 deficiency. Most notably, cells in which USP7 expression has been compromised are unable to efficiently promote elongation of nascent DNA synthesis following UV irradiation and are thus deficient in post-replication repair. These cells also exhibit reduced levels of PCNA monoubiquitylation and reduced recruitment of DNA polymerase h (one of the TLS polymerases) at sites of stalled replication. A consensus motif on RAD18, also present in other USP7-binding proteins,4 is vital for interaction with USP7 as well as for RAD18 stability. While mutation of this motif abolishes the interaction with USP7 and renders RAD18 susceptible to proteasomal degradation, this RAD18 mutant, if transiently expressed at levels comparable to the wild-type protein, is proficient at promoting TLS following UV irradiation. This suggests that tight control of RAD18 protein turnover by USP7 is as important for DDT as the regulation of its function (Fig. 1). Although many questions arise from our findings, the principle one is why there is a requirement for RAD18 to be protected from proteasomal degradation. Targeted polyubiquitylation and degradation of other ubiquitin ligases that are known substrates of USP7 has been reported to occur in a cell cycle-dependent manner, primarily at the G2/M transition.5,6 However, previous work has indicated that RAD18 protein levels show only limited fluctuation throughout the cell cycle.7 Interestingly, we noted that the unmodified form of Rad18 was more susceptible to degradation upon USP7 loss than the monoubiquitylated form.2 One plausible explanation could be differential cellular compartmentalization, as the monoubiquitylated form was previously shown to be primarily cytoplasmic.8 Another possibility is that the unmodified form of RAD18, previously suggested to be the active form,9 is targeted for polyubiquitylation and subsequent degradation unless it is protected via dimerization with monoubiquitylated RAD18 or is actively engaged in DDT. This could provide an elegant mechanism to suppress the aberrant use of DTT in the absence of DNA damage or could provide a way of inactivating DTT pathways when a lesion has been bypassed.
e1063571-2
A. ZLATANOU AND G.S. STEWART
Figure 1. USP7 is required for RAD18 stability and DNA damage tolerance. The interaction of RAD18 with USP7 prevents its poly-ubiquitylation and subsequent degradation. Maintenance of RAD18 pools in the cell promotes DNA damage tolerance (DDT) under conditions of genotoxic stress. DTT is a collective term for multiple pathways employed by the cell to ensure the completion of DNA synthesis in the presence of genetic lesions that obstruct replication fork progression. Following depletion or chemical inactivation of USP7, RAD18 becomes prone to poly-ubiquitylation and degradation by the proteasome. As a consequence, DDT is compromised, which in the presence of replication stress, leads to fork collapse, genome instability and ultimately cell death.
Nevertheless, it is clear from our data that fine-tuning RAD18 protein turnover might represent an additional way of regulating RAD18 activity. The significance of this is highlighted by the fact that while loss of RAD18 sensitizes cells to genotoxic agents, especially those that interfere with replication, elevated levels of the protein could also perturb DNA replication processivity by inappropriate PCNA ubiquitylation and TLS polymerase recruitment leading to increased mutagenesis. This is particularly relevant in light of the observation that primary melanoma tumors overexpress RAD18 and this correlates with reduced patient survival.10 Although the contribution of RAD18 overexpression to tumorigenesis is unclear, specifically targeting these tumor cells using recently developed USP7 inhibitors to compromise RAD18-dependent functions could be an additional therapeutic approach. To date, research investigating the therapeutic potential of inhibiting USP7 function has primarily focused on its involvement in regulating the p53 (TP53, best known as p53) pathway. However, our data indicate that USP7 inhibitors could be used to hypersensitize tumors, including those lacking functional p53, to chemotherapeutic agents that cause replication damage by compromising RAD18-dependent pathways. Based on this, it is clear that the role of USP7 in regulating the DNA damage response requires further investigation as it is likely to uncover unexpected pathways that can be exploited therapeutically.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References 1. Sale JE. Competition, collaboration and coordination–determining how cells bypass DNA damage. J Cell Sci 2012; 125:1633-43; PMID:22499669; http://dx.doi.org/10.1242/jcs.094748
2. Zlatanou A, Sabbioneda S, Miller ES, Greenwalt A, Aggathanggelou A, Maurice MM, Lehmann AR, Stankovic T, Reverdy C, Colland F, et al. USP7 is essential for maintaining Rad18 stability and DNA damage tolerance. Oncogene 2015 3. Nicholson B, Suresh Kumar KG. The multifaceted roles of USP7: new therapeutic opportunities. Cell Biochem Biophys 2011; 60:61-8; PMID:21468693; http://dx.doi.org/10.1007/s12013-011-9185-5 4. Sheng Y, Saridakis V, Sarkari F, Duan S, Wu T, Arrowsmith CH, Frappier L. Molecular recognition of p53 and MDM2 by USP7/ HAUSP. Nat Struct Mol Biol 2006; 13:285-91; PMID:16474402; http://dx.doi.org/10.1038/nsmb1067 5. Kim JS, Park YY, Park SY, Cho H, Kang D, Cho H. The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells. J Biol Chem 2011; 286:30615-23; PMID:21768102; http://dx.doi. org/10.1074/jbc.M111.231803 6. Ma H, Chen H, Guo X, Wang Z, Sowa ME, Zheng L, Hu S, Zeng P, Guo R, Diao J, et al. M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability. Proc Natl Acad Sci U S A 2012; 109:4828-33; PMID:22411829; http://dx.doi.org/10.1073/ pnas.1116349109 7. Masuyama S, Tateishi S, Yomogida K, Nishimune Y, Suzuki K, Sakuraba Y, Inoue H, Ogawa M, Yamaizumi M. Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions. Genes cell 2005; 10:753-62; http://dx.doi.org/10.1111/j.1365-2443.2005.00874.x 8. Miyase S, Tateishi S, Watanabe K, Tomita K, Suzuki K, Inoue H, Yamaizumi M. Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination. J Biol Chem 2005; 280:515-24; PMID:15509568; http://dx.doi.org/10.1074/jbc.M409219200 9. Zeman MK, Lin JR, Freire R, Cimprich KA. DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis. J Cell Biol 2014; 206:183-97; PMID:25023518; http://dx.doi.org/ 10.1083/jcb.201311063 10. Wong RP, Aguissa-Toure AH, Wani AA, Khosravi S, Martinka M, Li G. Elevated expression of Rad18 regulates melanoma cell proliferation. Pigment Cell Melanoma Res 2012; 25:213-8; PMID:22145991; http:// dx.doi.org/10.1111/j.1755-148X.2011.00948.x
AUTHOR’S VIEW
Damaged replication forks tolerate USP7 to maintain genome stability Anastasia Zlatanou and Grant S. Stewart School of Cancer Sciences, University of Birmingham, Birmingham, UK
ABSTRACT
ARTICLE HISTORY
RAD18 functions to promote DNA damage tolerance (DTT), a process that ensures faithful genome duplication. Protein ubiquitylation/deubiquitylation is a critical regulatory mechanism controlling DTT. Recently, we have identified the deubiquitylating enzyme USP7 as a component of the DTT machinery that acts to protect RAD18 from proteasome-dependent degradation.
Received 11 June 2015 Revised 15 June 2015 Accepted 15 June 2015
Obstacles that interfere with DNA replication can have detrimental consequences for genomic stability and cell viability. Replication fork progression can be hindered by the presence of bulky lesions because the replicative polymerases are unable to synthesize across a damaged DNA template. Cells employ a variety of mechanisms to tolerate and bypass these lesions, collectively designated DNA damage tolerance (DTT), in order to allow the completion of DNA synthesis. These include the recruitment of alternative polymerases that can synthesize across the lesion or a temporary switch of DNA synthesis to use the undamaged nascent DNA as a template.1 The E3 ubiquitin ligase RAD18 regulates both branches of DDT, primarily through its ability to monoubiquitylate proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity factor. This modification of PCNA facilitates the temporary replacement of replicative polymerases with trans-lesion synthesis (TLS) polymerases, which are able to replicate across the lesion albeit at the cost of increased mutagenesis due to their poor fidelity and lack of an exonuclease proofreading activity. Further ubiquitylation of PCNA by two other ubiquitin ligases that associate with RAD18— SHPRH and HLTF—promotes a less-characterized mode of lesion bypass that is error-free and is termed ‘template switching’. This pathway involves the replicative polymerase and uses the newly synthesized daughter strand as an undamaged DNA template in order to synthesize past the lesion. Polyubiquitylation, particularly with K48-linked ubiquitin chains, is primarily associated with targeting proteins for proteasome-dependent degradation. However, little is known about how it contributes to the control of DNA replication in the presence of potentially deleterious genetic damage. We have recently reported that RAD18 undergoes polyubiquitylation and proteasomal degradation unless it is protected by the catalytic activity of the deubiquitylating enzyme ubiquitin-specific peptidase 7 (USP7, also known as HAUSP).2 USP7 has been implicated in the regulation of multiple different cellular pathways including viral integration, DNA repair, apoptosis, and epigenetic transmission. This function is primarily mediated through its ability to regulate the stability of its interacting partners by dismantling polyubiquitin chains attached to them.3 Consistent with this, our work demonstrates that a
CONTACT Grant S. Stewart © 2016 Taylor & Francis Group, LLC
[email protected]
significant fraction of RAD18 becomes unstable in the absence of USP7 and, as a result, USP7-depleted cells phenocopy RAD18 deficiency. Most notably, cells in which USP7 expression has been compromised are unable to efficiently promote elongation of nascent DNA synthesis following UV irradiation and are thus deficient in post-replication repair. These cells also exhibit reduced levels of PCNA monoubiquitylation and reduced recruitment of DNA polymerase h (one of the TLS polymerases) at sites of stalled replication. A consensus motif on RAD18, also present in other USP7-binding proteins,4 is vital for interaction with USP7 as well as for RAD18 stability. While mutation of this motif abolishes the interaction with USP7 and renders RAD18 susceptible to proteasomal degradation, this RAD18 mutant, if transiently expressed at levels comparable to the wild-type protein, is proficient at promoting TLS following UV irradiation. This suggests that tight control of RAD18 protein turnover by USP7 is as important for DDT as the regulation of its function (Fig. 1). Although many questions arise from our findings, the principle one is why there is a requirement for RAD18 to be protected from proteasomal degradation. Targeted polyubiquitylation and degradation of other ubiquitin ligases that are known substrates of USP7 has been reported to occur in a cell cycle-dependent manner, primarily at the G2/M transition.5,6 However, previous work has indicated that RAD18 protein levels show only limited fluctuation throughout the cell cycle.7 Interestingly, we noted that the unmodified form of Rad18 was more susceptible to degradation upon USP7 loss than the monoubiquitylated form.2 One plausible explanation could be differential cellular compartmentalization, as the monoubiquitylated form was previously shown to be primarily cytoplasmic.8 Another possibility is that the unmodified form of RAD18, previously suggested to be the active form,9 is targeted for polyubiquitylation and subsequent degradation unless it is protected via dimerization with monoubiquitylated RAD18 or is actively engaged in DDT. This could provide an elegant mechanism to suppress the aberrant use of DTT in the absence of DNA damage or could provide a way of inactivating DTT pathways when a lesion has been bypassed.
e1063571-2
A. ZLATANOU AND G.S. STEWART
Figure 1. USP7 is required for RAD18 stability and DNA damage tolerance. The interaction of RAD18 with USP7 prevents its poly-ubiquitylation and subsequent degradation. Maintenance of RAD18 pools in the cell promotes DNA damage tolerance (DDT) under conditions of genotoxic stress. DTT is a collective term for multiple pathways employed by the cell to ensure the completion of DNA synthesis in the presence of genetic lesions that obstruct replication fork progression. Following depletion or chemical inactivation of USP7, RAD18 becomes prone to poly-ubiquitylation and degradation by the proteasome. As a consequence, DDT is compromised, which in the presence of replication stress, leads to fork collapse, genome instability and ultimately cell death.
Nevertheless, it is clear from our data that fine-tuning RAD18 protein turnover might represent an additional way of regulating RAD18 activity. The significance of this is highlighted by the fact that while loss of RAD18 sensitizes cells to genotoxic agents, especially those that interfere with replication, elevated levels of the protein could also perturb DNA replication processivity by inappropriate PCNA ubiquitylation and TLS polymerase recruitment leading to increased mutagenesis. This is particularly relevant in light of the observation that primary melanoma tumors overexpress RAD18 and this correlates with reduced patient survival.10 Although the contribution of RAD18 overexpression to tumorigenesis is unclear, specifically targeting these tumor cells using recently developed USP7 inhibitors to compromise RAD18-dependent functions could be an additional therapeutic approach. To date, research investigating the therapeutic potential of inhibiting USP7 function has primarily focused on its involvement in regulating the p53 (TP53, best known as p53) pathway. However, our data indicate that USP7 inhibitors could be used to hypersensitize tumors, including those lacking functional p53, to chemotherapeutic agents that cause replication damage by compromising RAD18-dependent pathways. Based on this, it is clear that the role of USP7 in regulating the DNA damage response requires further investigation as it is likely to uncover unexpected pathways that can be exploited therapeutically.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References 1. Sale JE. Competition, collaboration and coordination–determining how cells bypass DNA damage. J Cell Sci 2012; 125:1633-43; PMID:22499669; http://dx.doi.org/10.1242/jcs.094748
2. Zlatanou A, Sabbioneda S, Miller ES, Greenwalt A, Aggathanggelou A, Maurice MM, Lehmann AR, Stankovic T, Reverdy C, Colland F, et al. USP7 is essential for maintaining Rad18 stability and DNA damage tolerance. Oncogene 2015 3. Nicholson B, Suresh Kumar KG. The multifaceted roles of USP7: new therapeutic opportunities. Cell Biochem Biophys 2011; 60:61-8; PMID:21468693; http://dx.doi.org/10.1007/s12013-011-9185-5 4. Sheng Y, Saridakis V, Sarkari F, Duan S, Wu T, Arrowsmith CH, Frappier L. Molecular recognition of p53 and MDM2 by USP7/ HAUSP. Nat Struct Mol Biol 2006; 13:285-91; PMID:16474402; http://dx.doi.org/10.1038/nsmb1067 5. Kim JS, Park YY, Park SY, Cho H, Kang D, Cho H. The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells. J Biol Chem 2011; 286:30615-23; PMID:21768102; http://dx.doi. org/10.1074/jbc.M111.231803 6. Ma H, Chen H, Guo X, Wang Z, Sowa ME, Zheng L, Hu S, Zeng P, Guo R, Diao J, et al. M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability. Proc Natl Acad Sci U S A 2012; 109:4828-33; PMID:22411829; http://dx.doi.org/10.1073/ pnas.1116349109 7. Masuyama S, Tateishi S, Yomogida K, Nishimune Y, Suzuki K, Sakuraba Y, Inoue H, Ogawa M, Yamaizumi M. Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions. Genes cell 2005; 10:753-62; http://dx.doi.org/10.1111/j.1365-2443.2005.00874.x 8. Miyase S, Tateishi S, Watanabe K, Tomita K, Suzuki K, Inoue H, Yamaizumi M. Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination. J Biol Chem 2005; 280:515-24; PMID:15509568; http://dx.doi.org/10.1074/jbc.M409219200 9. Zeman MK, Lin JR, Freire R, Cimprich KA. DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis. J Cell Biol 2014; 206:183-97; PMID:25023518; http://dx.doi.org/ 10.1083/jcb.201311063 10. Wong RP, Aguissa-Toure AH, Wani AA, Khosravi S, Martinka M, Li G. Elevated expression of Rad18 regulates melanoma cell proliferation. Pigment Cell Melanoma Res 2012; 25:213-8; PMID:22145991; http:// dx.doi.org/10.1111/j.1755-148X.2011.00948.x
