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Regulation of base excision repair proteins by ubiquitylation
E XP ER I ME NTAL C E LL RE S E ARCH
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Available online at www.sciencedirect.com
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Review Article
Regulation of base excision repair proteins by ubiquitylation Matthew J. Edmonds, Jason L. Parsonsn North West Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK
article information
abstract
Article Chronology:
Human cellular DNA is under constant attack from both endogenous and exogenous mutagens,
Received 23 May 2014
and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged
Received in revised form
DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying
21 July 2014
complexity. BER thus maintains genome stability, and prevents the development of human
Accepted 24 July 2014
diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues
Keywords: Base excision repair DNA repair DNA damage
from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.
Ubiquitin
& 2014 Elsevier Inc. All rights reserved.
Ubiquitylation
Contents The cellular response to DNA base damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of BER components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Helix–hairpin–helix (HhH) DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endonuclease VIII-like (NEIL) DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uracil DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: AP, apurinic/apyrimidinic; APE1, AP endonuclease 1; ARF, alternate reading frame tumour suppressor protein; ATM, ataxia telangiectasia mutated; BER, base excision repair; CHIP, C-terminus of Hsc70-interacting protein; Cul, cullin E3 ligase; DDB1, DNA damage binding protein 1; dRP, deoxyribosephosphate; DUB, deubiquitylation enzyme; HhH, helix–hairpin–helix; Lig III, DNA ligase IIIα; MBD4, methyl CpG-binding domain protein 4; MDM2, mouse double minute 2; Mule, Mcl1 ubiquitin ligase E3; MutYH, MutY homologue; NEIL, endonuclease VIII-like; NTH1, endonuclease III homologue; OGG1, 8-oxoguanine DNA glycosylase; PNKP, polynucleotide kinase phosphatase; Pol β, DNA polymerase β; siRNA, small interfering RNA; SMUG1, single-strand-selective uracil-DNA glycosylase; STRAP, serine–threonine kinase receptor-associated protein; SUMO, small ubiquitin-like modifier; TDG, thymine-DNA glycosylase; UBR3, ubiquitin protein ligase E3 component n-recognin 3; UNG, uracil-DNA glycosylase; UPP, ubiquitin proteasome pathway; USP47, ubiquitin-specific protease 47; XRCC1, X-ray cross complementing protein 1. n
Corresponding author. E-mail address: [email protected] (J.L. Parsons).
http://dx.doi.org/10.1016/j.yexcr.2014.07.031 0014-4827/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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End processors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gap fillers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nick sealers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The cellular response to DNA base damage Human cells have been estimated to suffer around 10,000 DNA base lesions per cell per day from endogenous and environmental factors, which pose a major threat to the integrity of the human genome [1]. These DNA base lesions include sites of base loss (apurinic/apyrimidinic (AP) sites) and oxidised DNA bases, such as 8-oxoguanine (8-oxoG), which is known to be highly mutagenic. The base excision repair (BER) pathway actively repairs DNA base damage, therefore playing a vital role in genome stability, and thus in the prevention of human diseases, such as premature ageing, neurodegenerative diseases and cancer. BER is a highly co-ordinated process aimed at the rapid repair of these simple DNA base lesions (Fig. 1). The initial damage recognition step is performed by DNA glycosylases, which scan along the DNA backbone checking for damaged bases using a ‘base-flipping’ mechanism [2,3]. There are 11 known human DNA glycosylases, each with their own specificity for particular base
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lesions. When a lesion is found, the DNA glycosylase cleaves the N-glycosidic bond between the base and the phosphodiester DNA backbone to excise the damaged base. The resulting AP site is processed by AP endonuclease 1 (APE1) to leave a one nucleotide gap flanked by 30 -hydroxyl and 50 -deoxyribosephosphate (50 -dRP) ends [4,5]. This site is further processed by DNA polymerase β (Pol β) which removes the 50 -dRP moiety and simultaneously inserts a new, correct nucleotide into the repair gap [6,7]. The remaining single-strand nick is finally sealed by a complex of DNA ligase IIIα (Lig III) and X-ray cross-complementing protein 1 (XRCC1) [8,9]. This is commonly known as the short-patch BER pathway (Fig. 1, left pathway), through which the majority of damaged DNA bases are repaired [10]. Interestingly, the endonuclease VIII-like proteins (NEIL1–3) were discovered over a decade ago to proceed via an APE1-independent pathway [3,11]. These enzymes generate a single nucleotide gap flanked by 50 - and 30 -phosphate ends that require processing by the 30 -phosphatase activity of polynucleotide kinase phosphatase (PNKP) prior to subsequent Pol β and
β,δ-elimination
DNA glycosylase damaged base excision P
APE1
end processing
dRP
P
PNKP P
DNA polymerase β gap filling
DNA ligase IIIα/XRCC1 nick sealing
Fig. 1 – Repair of DNA base lesions by the BER pathway. Damage-specific DNA glycosylases (such as OGG1, NTH1 and UNG) excise the damaged base by cleavage of the N-glycosidic bond leaving an AP site. This is incised by APE1 to create a single strand break containing a 50 -dRP moiety, which is removed by the dRP lyase activity of Pol β that simultaneously fills the gap with a new nucleotide (left branch). In contrast, BER initiated by the NEIL DNA glycosylases (NEIL1, 2 and 3) possess a β,δ-elimination activity which creates a single nucleotide gap containing 30 - and 50 -phosphate ends. The 30 -phosphate is removed by PNKP before nucleotide insertion by Pol β (right branch). Finally, the remaining nick in the phosphodiester backbone is sealed by Lig III-XRCC1 complex to complete BER. Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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XRCC1-Lig III activity (Fig. 1, right pathway). Nevertheless, the contribution of NEIL enzymes to cellular BER activity is still not fully understood.
Table 1 – Summary of the known E3 ubiquitin ligases for the major BER proteins. E3 ligase
BER protein
Citation
CHIP
OGG1 Pol β XRCC1 Lig III UNG SMUG1 UNG SMUG1 PNKP (with STRAP) XRCC1 Lig III APE1 MutYH Pol β APE1 NTH1 NEIL2 NEIL3 MBD4 TDG NEIL1
[14] [41] [41] [41] [27] [27] [27] [27] [38]
Regulation of BER components Regulation of the BER pathway is essential in allowing mammalian cells to respond appropriately to changes in endogenous DNA damage levels, and it has become evident over the last 10 years that the predominant mechanism through which this tight control is achieved is through post-translational modifications, such as acetylation and phosphorylation, of BER proteins [12]. Indeed there is a growing body of evidence suggesting that the subcellular levels of BER proteins are regulated by alteration of protein stability and localisation mediated by ubiquitylation and the ubiquitin-proteasome pathway (UPP). Ubiquitin is a small 76 amino acid, approximately 8 kDa protein, which is generally attached to specific lysine residues found in target proteins. This is performed in an ATP-dependent manner by an enzyme cascade initiated by an E1 activating enzyme, followed by a conjugating E2 enzyme and finally by an E3 ubiquitin ligase specific for the substrate protein. Currently, there are over 600 E3 ubiquitin ligases proposed to exist, although the substrate specificity of most has not been fully examined. Proteins may be modified by single ubiquitin moieties, termed monoubiquitylation, or by multiple ubiquitin chains, termed polyubiquitylation, and it is typically the latter in which chains are formed through internal lysine 48 residues of ubiquitin which targets substrates for degradation by the UPP [13]. Conversely, monoubiquitylation is thought to contribute predominantly to cell signalling functions of modified proteins, for example regulation of their cellular activity or subcellular localisation. Below, we will summarise some of the accumulating evidence that key BER proteins are regulated through ubiquitylation (see also Table 1), and also that cellular BER protein levels can be controlled by the UPP, which modulates the cellular response to DNA damage.
DNA glycosylases There is accumulating evidence that many of the damage-specific DNA glycosylases are ubiquitylated, although the E3 ubiquitin ligase enzymes responsible and the functional consequences remain largely unexplored.
Helix–hairpin–helix (HhH) DNA glycosylases The HhH glycosylases are named for the eponymous structural motif they share and two members of the human family are 8-oxoguanine DNA glycosylase (OGG1) and endonuclease III homologue (NTH1). OGG1, which excises 8-oxoguanine lesions, has not been identified in proteome-wide ubiquitylation screens, although it has been discovered to be ubiquitylated by the E3 ubiquitin ligase C-terminus of Hsc70-interacting protein (CHIP) [14]. Indeed, OGG1 was found to be degraded specifically in response to mild hyperthermia, which led to a reduced repair capacity and a cell growth defect in HeLa cells in which the experiments were performed. Undegraded OGG1 was also observed to relocalise from the nucleus to a perinuclear region, further reducing repair capacity. In contrast, ubiquitylation of
3
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Cul1 Cul4
Iduna/RNF146 MDM2 Mule UBR3 Unknown, identified in ubiquitylation screens
Unknown, no evidence of ubiquitylation
[46] [46] [32] [16] [42] [33] [15,22– 24,26]
NTH1, a DNA glycosylase whose substrates include oxidised pyrimidines such as 5-hydroxycytosine and thymine glycol, has only been described in a proteomic screen [15] and has not been explored further. Nevertheless, the importance of using proteomic screens only as a guide to potential substrates should be kept in mind, as their inherent limitations lead to the possible existence of both false positives and genuine candidates which may be below the detection threshold. The other member of the HhH glycosylase family is the MutY homologue (MutYH), which excises predominantly adenine residues that have been incorrectly incorporated opposite 8-oxoguanine residues following DNA replication or transcription. MutYH has been shown to be ubiquitylated in vitro and in vivo by the E3 ubiquitin ligase, Mcl1 ubiquitin ligase E3/ARF (alternate reading frame tumour supressor)-binding protein 1 (Mule/ARFBP1) between residues 475 and 500, targeting it for proteasomal degradation [16]. This ubiquitylation was also shown to regulate the subcellular distribution of MutYH. Indeed, inhibiting ubiquitylation of MutYH using a mutant form of the protein with lysine residues mutated to arginines in the ubiquitylation region caused the protein to accumulate in a chromatin-bound fraction [16]. Overexpression of Mule in ovarian cancer A2780 cells gave them a greater mutational frequency in response to oxidative stress, although it was not tested whether this was specifically due to increased MutYH degradation [16]. As will become evident later in this review, other members of the BER pathway are known to be substrates for ubiquitylation by Mule, so it is likely they may also play a role in this effect. Interestingly, MutYH and APE1 have previously been shown to physically interact in a complex which is stabilised by the Rad9–Rad1–Hus1 complex, a checkpoint clamp, allowing immediate processing of the AP site created by MutYH activity on a damaged base [17]. Although the binding interface is away from the region ubiquitylated by Mule [16,17], the localisation of ubiquitylated MutYH away from the nucleus
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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gives scope for indirect regulation of the DNA glycosylase with APE1. This suggests that specific E3 ubiquitin ligases, and possibly deubiquitylation enzymes (DUBs), may be able to respond to cellular stresses, resulting in the modulation of ubiquitylated forms of DNA glycosylases, such as MutYH, and subsequently their cellular localisation. This would regulate their interaction with DNA, or in the case of MutYH with cellular proteins such as APE1 and/or Rad9–Rad1–Hus1, and ultimately regulate their involvement in the DNA damage repair response.
Endonuclease VIII-like (NEIL) DNA glycosylases The NEIL DNA glycosylase family possess a similar range of substrate specificity to the HhH glycosylases OGG1 and NTH1, but comprise a separate family due to their closer structural homology to the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) [2,3]. There are three members of this family, namely NEIL1–3, and there has been evidence that in fact these DNA glycosylases may be involved in the repair of specific types of DNA base damage, such as those in close proximity to another DNA lesion [18,19], in single stranded DNA or bubble structures mimicking those generated during DNA transcription and replication [20], or in telomeric DNA [21]. While NEIL2 and NEIL3 (but not NEIL1) have been shown to be ubiquitylated in proteomic screens [22–24], these studies have not been taken any further. Interestingly, NEIL2 has been shown to undergo acetylation by p300 principally at two lysine residues, namely on lysine 49, which abrogates its base excision and AP lyase activities, and secondly on lysine 153, which has no effect on its enzymatic activities [25]. It can therefore be speculated that there is a potential for overlapping sites of ubiquitylation and acetylation on NEIL2; thus revealing multiple levels of regulation. It is possible that acetylation, in addition to modulating enzymatic activity, may inhibit ubiquitylation-dependent proteasomal degradation and therefore maintain the cellular levels of the protein in an inactive state prior to its requirement for DNA repair.
Uracil DNA glycosylases All four members of the uracil DNA glycosylase family, namely uracil-DNA glycosylase (UNG), single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), methyl-CpG binding domain protein 4 (MBD4) and thymine-DNA glycosylase (TDG), have been repeatedly identified in proteomic screens for ubiquitylated proteins [15,22–24,26]. However, further research has been limited to UNG and SMUG1, whose ubiquitylation by the E3 ligases cullin 1 (Cul1) and cullin 4 (Cul4) and subsequent proteasomal degradation has been found to be stimulated by binding to the human immunodeficiency virus (HIV) accessory protein viral protein R (Vpr) [27]. This is thought to be a mechanism for reducing the number of AP sites in viral reverse transcripts, as a consequence of UNG and SMUG1 activity on uracil residues generated by the cellular enzyme apolipoprotein B mRNA editing enzyme 3 (APOBEC3), and therefore enhancing viral replication. There is a body of evidence suggesting that modification of TDG with the small ubiquitin-like modifier (SUMO)-1, -2 and -3 at lysine 330, located in the C-terminal region may be important for its activity [28–31]. Indeed, a non-covalent interaction of SUMO with this C-terminal region of TDG reduces its AP site binding affinity [28] by altering the conformation of the protein, thereby competitively inhibiting its DNA-binding domain [30,31]. Given
] (]]]]) ]]]–]]]
that both ubiquitylation and SUMOylation occur on lysine residues, it would be interesting to investigate whether the respective sites on TDG overlap or are distinct. Consequently, the two modifications may work in parallel whereby SUMO alters its AP site affinity to promote dissociation from the DNA after the damaged base has been excised, and the addition of ubiquitin would target TDG for proteasomal degradation.
End processors APE1 is the major AP endonuclease activity employed during BER. The first evidence for the existence of an E3 ubiquitin ligase regulating APE1 was the observation that following DNA damage, APE1 was polyubiquitylated on lysines 24, 25 and 27 by the mouse double minute 2 (MDM2) enzyme [32]. MDM2 is more commonly known to regulate protein levels of the p53 tumour suppressor protein that acts as a transcription factor mediating the cellular response to genotoxic stress. Indeed, APE1 polyubiquitylation was found to be dependent on the presence of p53. However, these data have been superseded by evidence that the major E3 ubiquitin ligase activity for APE1 is ubiquitin protein ligase E3 component n-recognin 3 (UBR3), and not MDM2 [33]. This was discovered by employing APE1 as a substrate in an in vitro ubiquitylation assay using fractionated HeLa cell extracts generated by column chromatography. However, similar to the first study, UBR3 polyubiquitylated APE1 within its N-terminus, and specifically this was spread over nine lysine residues within the first 35 amino acids on the protein. Key evidence for demonstrating that UBR3 is a key regulator of APE1 protein levels was provided by the fact that mouse embryonic fibroblasts (MEFs) lacking the Ubr3 gene not only have increased abundance of APE1 as a result of the lack of ubiquitylation-dependent degradation, but also that these cells showed signs of genetic instability as evidenced by increased DNA double strand break formation [33]. Consequently, it was suggested that an imbalance in the level of APE1, in comparison to the downstream enzyme Pol β, causes a build-up of DNA single strand breaks and that when cells progress through DNA replication, these lesions induce replication fork stalling leading to double strand break formation. It is interesting to note that acetylation has been observed on lysine residues in the same unstructured N-terminal region of APE1 which is ubiquitylated [34]. Acetylation of these residues alters the structure of this region and leads to binding of the deacetylase sirtuin 1, which causes deacetylation of APE1, and is thought to promote BER by increasing binding of the protein to XRCC1 and enhancing AP endonuclease activity [35]. As the N-terminal region has been proposed to mediate the interaction between APE1 and both rRNA and the nucleolar protein nucleophosmin, it would be interesting to investigate the effects of cross-talk between ubiquitylation and acetylation on the enzymatic activity and protein levels of APE1, and the consequences for the efficiency of BER and genome stability. BER initiated by the NEIL DNA glycosylases generates a single nucleotide gap containing a 30 -phosphate end which requires further processing by PNKP to allow gap filling and ligation to proceed (Fig. 1). Recently, an interplay between phosphorylation and ubiquitylation of PNKP has been uncovered which has been shown to regulate cellular PNKP protein levels in response to DNA damage. While PNKP phosphorylation on serines 114 and 126 had previously been shown to be mediated by the protein kinase,
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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ataxia telangiectasia mutated (ATM) and to regulate DNA double strand break repair [36,37], this site-specific PNKP phosphorylation was also observed in HCT116p53þ/þ cells in response to oxidative stress [38]. Concurrent with this ATM-dependent phosphorylation was an approximate 1.5-fold increase in PNKP protein levels peaking at 4–6 h post-treatment which correlated with a decrease in PNKP polyubiquitylation. The E3 ubiquitin ligase targeting PNKP for ubiquitylation was consequently purified from HeLa cell extracts and found to consist of a complex containing Cul4A, DNA damage binding protein 1 (DDB1) and a WD-40 repeat protein called serine–threonine kinase receptor-associated protein (STRAP). WD-40 repeat proteins, such as STRAP, are the adaptor proteins that interact with DDB1 and which provide specificity of the multiple Cul4A–DDB1 associated E3 ubiquitin ligase complexes to substrate proteins [39]. The sites of PNKP ubiquitylation by Cul4A–DDB1–STRAP were found to be lysines 414, 417 and 484 and Strap knockout MEFs demonstrated an increase in PNKP protein levels, and resistance to oxidative stress [38]. Furthermore, the link between ATM-dependent phosphorylation and inhibition of Cul4A–DDB1–STRAP dependent ubiquitylation was provided by evidence that a phosphomimetic S114/ 126E PNKP mutant displayed reduced ubiquitylation by Cul4A– DDB1–STRAP in vitro and in vivo, and consequently the protein was more stable when introduced into HCT116p53þ/þ cells. This suggests that phosphorylation of PNKP by ATM is directly responsible for the stabilisation of PNKP in response to oxidative stress, and provides a potential explanation for the increased sensitivity of ATM deficient cells to oxidative stress [40].
Gap fillers The stability of Pol β, the major DNA polymerase employed during BER, has been discovered to be dependent on the presence of XRCC1 on damaged DNA [41]. Consequently, using Pol β as a substrate in an in vitro ubiquitylation system combined with fractionated HeLa cell extracts generated by column chromatography, the major E3 ubiquitin ligase targeting Pol β for polyubiquitylation was shown to be the E3 ubiquitin ligase CHIP, and that the site was localised to the 8 kDa N-terminal domain of Pol β [41]. Indeed, overexpression of CHIP in HeLa cells decreased cellular Pol β protein levels, and conversely, small interfering RNA (siRNA) knockdown of CHIP increased protein levels. Subsequent to this study, another E3 ubiquitin ligase activity for Pol β was discovered, this time supporting monoubiquitylation [42]. Monoubiquitylation was found to occur on lysines 41, 61 and 81 by the E3 ubiquitin ligase Mule/ARF-BP1, which compartmentalised the protein to the cytoplasm. In fact, monoubiquitylation on these site-specific residues of Pol β was found to be a prerequisite for subsequent polyubiquitylation by CHIP, as shown by modulation in monoubiquitylated Pol β protein levels following CHIP overexpression or siRNA knockdown in HeLa cells. The E3 ubiquitin ligase activity of Mule/ARF-BP1 is known to be inhibited by the tumour suppressor protein ARF [43], and consequently siRNA knockdown of ARF in HeLa cells caused an increase in Pol β monoubiquitylation [42]. Regulation of Pol β protein levels by Mule and ARF was furthermore shown to alter the proficiency of HeLa cells to undergo DNA damage repair induced by oxidative stress. Since ubiquitylation of proteins is a reversible modification, it was further predicted that a DUB capable of recycling Pol β from the monoubiquitylated pool contained within the cytoplasm may
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exist, which would allow cellular Pol β protein levels to adjust to the DNA damage environment. Indeed, using ubiquitylated Pol β as a substrate in an in vitro deubiquitylation system combined with fractionated HeLa cell extracts generated by column chromatography, the ubiquitin-specific protease 47 (USP47) was purified as the major DUB activity for Pol β [44]. DUB activity was confirmed in vitro using recombinant USP47 expressed and purified from bacterial cells, which interestingly was able to deubiquitylate both monoubiquitylated Pol β (generated by Mule) and polyubiquitylated Pol β (generated by CHIP). It was further discovered that USP47 regulates the cellular levels of Pol β, since siRNA knockdown of USP47 in HeLa cells resulted in an increase in monoubiquitylated Pol β levels found in the cytoplasm, and as a consequence caused a reduction in the cytoplasmic levels of the native protein. Interestingly, a simultaneous siRNA knockdown of Mule and USP47 was discovered to be able to restore exogenously expressed Pol β levels that were reduced by a USP47 siRNA knockdown alone, demonstrating that Mule was specifically targeting newly synthesised Pol β for ubiquitylation-dependent degradation. As a consequence of USP47-dependent regulation of cellular Pol β levels, a delay in the rate of repair of hydrogen peroxide and methyl methanesulfonate (MMS)-induced DNA damage, as well as reduced cell survival following hydrogen peroxide treatment, was observed in HeLa cells lacking USP47. The dRP lyase activity of Pol β is known to be mediated by Schiffbase formation using lysine 72, which has also previously been shown to be negatively regulated by acetylation by p300 [45]. Given the close proximity of this residue to those monoubiquitylated by Mule on lysines 41, 61 and 81, there is potential for crosstalk between these two regulatory modifications despite these appearing to occur in separate compartments of the cell. Nevertheless, it would be interesting to examine this proposed crosstalk, particularly during the DNA damage response. These studies have collectively demonstrated an important role for CHIP, Mule and USP47 in the regulation of cellular Pol β protein levels, and consequently in the cellular response to DNA damage. However, this mechanism appears to be calibrated to allow small changes in Pol β protein levels, presumably as a consequence of minor fluctuations in endogenous DNA damage generated through cellular metabolism. Therefore any significant, sustained elevations in DNA damage through exogenous stress which exceed cellular DNA repair capacity are likely to drive the cell into apoptosis.
Nick sealers The scaffold protein XRCC1 is in a stable complex with Lig III and the importance of cellular XRCC1 protein levels is highlighted by the fact that XRCC1 deficient cells contain reduced levels of Lig III, but also Pol β, and display hypersensitivity to DNA damaging agents [41,47]. Both XRCC1 and Lig III have been shown to be polyubiquitylated in vitro by CHIP, the same E3 ubiquitin ligase responsible for polyubiquitylation of Pol β [41]. Furthermore, knockdown of CHIP in HeLa cells using siRNA increased the stability of both XRCC1 and Lig III, showing CHIP-dependent ubiquitylation results in their degradation by the UPP. The site of ubiquitylation of XRCC1 was localised to the C-terminal region of the protein containing a BRCA1 C-terminus (BRCT) motif, since a truncated form of the protein lacking this region showed increased stability in comparison to the wild-type protein when transfected into XRCC1-deficient cells [41].
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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Both XRCC1 and Lig III are also subject to ubiquitylation by another E3 ubiquitin ligase activity which was identified as Iduna, or ring finger protein 146 (RNF146), although this is dependent on XRCC1 and Lig III being modified by poly(ADP-ribosyl)ation beforehand [46]. Iduna was found to protect against cell death induced by ionising radiation or treatment with the alkylating agent N-methylN-nitro-N-nitrosoguanidine (MNNG), although due to the number of targets identified for Iduna-dependent ubiquitylation, it is unclear whether this effect is mediated predominantly through XRCC1 and Lig III, and therefore requires further study.
Future perspectives The study of the regulation of components of the BER pathway, particularly through the UPP, is still at a very early stage and therefore the E3 ubiquitin ligases, the DUBs and consequently the mechanisms controlling BER proteins are still unclear. In particular, little is known about the regulation of the enzymes performing the initial step of BER, the DNA glycosylases, although there is evidence from proteomic screens that they are targets for ubiquitylation. It will be interesting to see in the future whether common themes emerge, for example, whether particular classes of BER enzymes are regulated by the same E3 ubiquitin ligases/ DUBs, or whether each BER component has its own specific complement of ubiquitylation/deubiquitylation enzyme(s). Interestingly, misregulation of BER protein levels has been observed in a range of human diseases, particularly in cancer but also in premature ageing and neurodegenerative diseases. Therefore, once uncovered, the next step would be to investigate the role of E3 ubiquitin ligases/DUBs targeting BER proteins, for their involvement in BER misregulation found in cells and tissues derived from patients with these diseases. Consequently, this detailed knowledge may help to uncover novel targets for drugs or small molecule inhibitors, which when combined with radiotherapy and/or chemotherapy could enhance current treatment regimens for patients with diseases containing altered BER protein expression.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
Acknowledgement
[18]
Jason Parsons is supported by funding from North West Cancer Research Fund (CR972 and CR1016) and by the Medical Research Council via a New Investigator Research Grant (MR/M000354/1).
[19]
[20]
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] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
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Review Article
Regulation of base excision repair proteins by ubiquitylation Matthew J. Edmonds, Jason L. Parsonsn North West Cancer Research Centre, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, 200 London Road, Liverpool L3 9TA, UK
article information
abstract
Article Chronology:
Human cellular DNA is under constant attack from both endogenous and exogenous mutagens,
Received 23 May 2014
and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged
Received in revised form
DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying
21 July 2014
complexity. BER thus maintains genome stability, and prevents the development of human
Accepted 24 July 2014
diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues
Keywords: Base excision repair DNA repair DNA damage
from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.
Ubiquitin
& 2014 Elsevier Inc. All rights reserved.
Ubiquitylation
Contents The cellular response to DNA base damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of BER components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Helix–hairpin–helix (HhH) DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endonuclease VIII-like (NEIL) DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uracil DNA glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 3 3 3 4 4
Abbreviations: AP, apurinic/apyrimidinic; APE1, AP endonuclease 1; ARF, alternate reading frame tumour suppressor protein; ATM, ataxia telangiectasia mutated; BER, base excision repair; CHIP, C-terminus of Hsc70-interacting protein; Cul, cullin E3 ligase; DDB1, DNA damage binding protein 1; dRP, deoxyribosephosphate; DUB, deubiquitylation enzyme; HhH, helix–hairpin–helix; Lig III, DNA ligase IIIα; MBD4, methyl CpG-binding domain protein 4; MDM2, mouse double minute 2; Mule, Mcl1 ubiquitin ligase E3; MutYH, MutY homologue; NEIL, endonuclease VIII-like; NTH1, endonuclease III homologue; OGG1, 8-oxoguanine DNA glycosylase; PNKP, polynucleotide kinase phosphatase; Pol β, DNA polymerase β; siRNA, small interfering RNA; SMUG1, single-strand-selective uracil-DNA glycosylase; STRAP, serine–threonine kinase receptor-associated protein; SUMO, small ubiquitin-like modifier; TDG, thymine-DNA glycosylase; UBR3, ubiquitin protein ligase E3 component n-recognin 3; UNG, uracil-DNA glycosylase; UPP, ubiquitin proteasome pathway; USP47, ubiquitin-specific protease 47; XRCC1, X-ray cross complementing protein 1. n
Corresponding author. E-mail address: [email protected] (J.L. Parsons).
http://dx.doi.org/10.1016/j.yexcr.2014.07.031 0014-4827/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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End processors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gap fillers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nick sealers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The cellular response to DNA base damage Human cells have been estimated to suffer around 10,000 DNA base lesions per cell per day from endogenous and environmental factors, which pose a major threat to the integrity of the human genome [1]. These DNA base lesions include sites of base loss (apurinic/apyrimidinic (AP) sites) and oxidised DNA bases, such as 8-oxoguanine (8-oxoG), which is known to be highly mutagenic. The base excision repair (BER) pathway actively repairs DNA base damage, therefore playing a vital role in genome stability, and thus in the prevention of human diseases, such as premature ageing, neurodegenerative diseases and cancer. BER is a highly co-ordinated process aimed at the rapid repair of these simple DNA base lesions (Fig. 1). The initial damage recognition step is performed by DNA glycosylases, which scan along the DNA backbone checking for damaged bases using a ‘base-flipping’ mechanism [2,3]. There are 11 known human DNA glycosylases, each with their own specificity for particular base
4 5 5 6 6 6
lesions. When a lesion is found, the DNA glycosylase cleaves the N-glycosidic bond between the base and the phosphodiester DNA backbone to excise the damaged base. The resulting AP site is processed by AP endonuclease 1 (APE1) to leave a one nucleotide gap flanked by 30 -hydroxyl and 50 -deoxyribosephosphate (50 -dRP) ends [4,5]. This site is further processed by DNA polymerase β (Pol β) which removes the 50 -dRP moiety and simultaneously inserts a new, correct nucleotide into the repair gap [6,7]. The remaining single-strand nick is finally sealed by a complex of DNA ligase IIIα (Lig III) and X-ray cross-complementing protein 1 (XRCC1) [8,9]. This is commonly known as the short-patch BER pathway (Fig. 1, left pathway), through which the majority of damaged DNA bases are repaired [10]. Interestingly, the endonuclease VIII-like proteins (NEIL1–3) were discovered over a decade ago to proceed via an APE1-independent pathway [3,11]. These enzymes generate a single nucleotide gap flanked by 50 - and 30 -phosphate ends that require processing by the 30 -phosphatase activity of polynucleotide kinase phosphatase (PNKP) prior to subsequent Pol β and
β,δ-elimination
DNA glycosylase damaged base excision P
APE1
end processing
dRP
P
PNKP P
DNA polymerase β gap filling
DNA ligase IIIα/XRCC1 nick sealing
Fig. 1 – Repair of DNA base lesions by the BER pathway. Damage-specific DNA glycosylases (such as OGG1, NTH1 and UNG) excise the damaged base by cleavage of the N-glycosidic bond leaving an AP site. This is incised by APE1 to create a single strand break containing a 50 -dRP moiety, which is removed by the dRP lyase activity of Pol β that simultaneously fills the gap with a new nucleotide (left branch). In contrast, BER initiated by the NEIL DNA glycosylases (NEIL1, 2 and 3) possess a β,δ-elimination activity which creates a single nucleotide gap containing 30 - and 50 -phosphate ends. The 30 -phosphate is removed by PNKP before nucleotide insertion by Pol β (right branch). Finally, the remaining nick in the phosphodiester backbone is sealed by Lig III-XRCC1 complex to complete BER. Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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XRCC1-Lig III activity (Fig. 1, right pathway). Nevertheless, the contribution of NEIL enzymes to cellular BER activity is still not fully understood.
Table 1 – Summary of the known E3 ubiquitin ligases for the major BER proteins. E3 ligase
BER protein
Citation
CHIP
OGG1 Pol β XRCC1 Lig III UNG SMUG1 UNG SMUG1 PNKP (with STRAP) XRCC1 Lig III APE1 MutYH Pol β APE1 NTH1 NEIL2 NEIL3 MBD4 TDG NEIL1
[14] [41] [41] [41] [27] [27] [27] [27] [38]
Regulation of BER components Regulation of the BER pathway is essential in allowing mammalian cells to respond appropriately to changes in endogenous DNA damage levels, and it has become evident over the last 10 years that the predominant mechanism through which this tight control is achieved is through post-translational modifications, such as acetylation and phosphorylation, of BER proteins [12]. Indeed there is a growing body of evidence suggesting that the subcellular levels of BER proteins are regulated by alteration of protein stability and localisation mediated by ubiquitylation and the ubiquitin-proteasome pathway (UPP). Ubiquitin is a small 76 amino acid, approximately 8 kDa protein, which is generally attached to specific lysine residues found in target proteins. This is performed in an ATP-dependent manner by an enzyme cascade initiated by an E1 activating enzyme, followed by a conjugating E2 enzyme and finally by an E3 ubiquitin ligase specific for the substrate protein. Currently, there are over 600 E3 ubiquitin ligases proposed to exist, although the substrate specificity of most has not been fully examined. Proteins may be modified by single ubiquitin moieties, termed monoubiquitylation, or by multiple ubiquitin chains, termed polyubiquitylation, and it is typically the latter in which chains are formed through internal lysine 48 residues of ubiquitin which targets substrates for degradation by the UPP [13]. Conversely, monoubiquitylation is thought to contribute predominantly to cell signalling functions of modified proteins, for example regulation of their cellular activity or subcellular localisation. Below, we will summarise some of the accumulating evidence that key BER proteins are regulated through ubiquitylation (see also Table 1), and also that cellular BER protein levels can be controlled by the UPP, which modulates the cellular response to DNA damage.
DNA glycosylases There is accumulating evidence that many of the damage-specific DNA glycosylases are ubiquitylated, although the E3 ubiquitin ligase enzymes responsible and the functional consequences remain largely unexplored.
Helix–hairpin–helix (HhH) DNA glycosylases The HhH glycosylases are named for the eponymous structural motif they share and two members of the human family are 8-oxoguanine DNA glycosylase (OGG1) and endonuclease III homologue (NTH1). OGG1, which excises 8-oxoguanine lesions, has not been identified in proteome-wide ubiquitylation screens, although it has been discovered to be ubiquitylated by the E3 ubiquitin ligase C-terminus of Hsc70-interacting protein (CHIP) [14]. Indeed, OGG1 was found to be degraded specifically in response to mild hyperthermia, which led to a reduced repair capacity and a cell growth defect in HeLa cells in which the experiments were performed. Undegraded OGG1 was also observed to relocalise from the nucleus to a perinuclear region, further reducing repair capacity. In contrast, ubiquitylation of
3
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Cul1 Cul4
Iduna/RNF146 MDM2 Mule UBR3 Unknown, identified in ubiquitylation screens
Unknown, no evidence of ubiquitylation
[46] [46] [32] [16] [42] [33] [15,22– 24,26]
NTH1, a DNA glycosylase whose substrates include oxidised pyrimidines such as 5-hydroxycytosine and thymine glycol, has only been described in a proteomic screen [15] and has not been explored further. Nevertheless, the importance of using proteomic screens only as a guide to potential substrates should be kept in mind, as their inherent limitations lead to the possible existence of both false positives and genuine candidates which may be below the detection threshold. The other member of the HhH glycosylase family is the MutY homologue (MutYH), which excises predominantly adenine residues that have been incorrectly incorporated opposite 8-oxoguanine residues following DNA replication or transcription. MutYH has been shown to be ubiquitylated in vitro and in vivo by the E3 ubiquitin ligase, Mcl1 ubiquitin ligase E3/ARF (alternate reading frame tumour supressor)-binding protein 1 (Mule/ARFBP1) between residues 475 and 500, targeting it for proteasomal degradation [16]. This ubiquitylation was also shown to regulate the subcellular distribution of MutYH. Indeed, inhibiting ubiquitylation of MutYH using a mutant form of the protein with lysine residues mutated to arginines in the ubiquitylation region caused the protein to accumulate in a chromatin-bound fraction [16]. Overexpression of Mule in ovarian cancer A2780 cells gave them a greater mutational frequency in response to oxidative stress, although it was not tested whether this was specifically due to increased MutYH degradation [16]. As will become evident later in this review, other members of the BER pathway are known to be substrates for ubiquitylation by Mule, so it is likely they may also play a role in this effect. Interestingly, MutYH and APE1 have previously been shown to physically interact in a complex which is stabilised by the Rad9–Rad1–Hus1 complex, a checkpoint clamp, allowing immediate processing of the AP site created by MutYH activity on a damaged base [17]. Although the binding interface is away from the region ubiquitylated by Mule [16,17], the localisation of ubiquitylated MutYH away from the nucleus
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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gives scope for indirect regulation of the DNA glycosylase with APE1. This suggests that specific E3 ubiquitin ligases, and possibly deubiquitylation enzymes (DUBs), may be able to respond to cellular stresses, resulting in the modulation of ubiquitylated forms of DNA glycosylases, such as MutYH, and subsequently their cellular localisation. This would regulate their interaction with DNA, or in the case of MutYH with cellular proteins such as APE1 and/or Rad9–Rad1–Hus1, and ultimately regulate their involvement in the DNA damage repair response.
Endonuclease VIII-like (NEIL) DNA glycosylases The NEIL DNA glycosylase family possess a similar range of substrate specificity to the HhH glycosylases OGG1 and NTH1, but comprise a separate family due to their closer structural homology to the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) [2,3]. There are three members of this family, namely NEIL1–3, and there has been evidence that in fact these DNA glycosylases may be involved in the repair of specific types of DNA base damage, such as those in close proximity to another DNA lesion [18,19], in single stranded DNA or bubble structures mimicking those generated during DNA transcription and replication [20], or in telomeric DNA [21]. While NEIL2 and NEIL3 (but not NEIL1) have been shown to be ubiquitylated in proteomic screens [22–24], these studies have not been taken any further. Interestingly, NEIL2 has been shown to undergo acetylation by p300 principally at two lysine residues, namely on lysine 49, which abrogates its base excision and AP lyase activities, and secondly on lysine 153, which has no effect on its enzymatic activities [25]. It can therefore be speculated that there is a potential for overlapping sites of ubiquitylation and acetylation on NEIL2; thus revealing multiple levels of regulation. It is possible that acetylation, in addition to modulating enzymatic activity, may inhibit ubiquitylation-dependent proteasomal degradation and therefore maintain the cellular levels of the protein in an inactive state prior to its requirement for DNA repair.
Uracil DNA glycosylases All four members of the uracil DNA glycosylase family, namely uracil-DNA glycosylase (UNG), single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), methyl-CpG binding domain protein 4 (MBD4) and thymine-DNA glycosylase (TDG), have been repeatedly identified in proteomic screens for ubiquitylated proteins [15,22–24,26]. However, further research has been limited to UNG and SMUG1, whose ubiquitylation by the E3 ligases cullin 1 (Cul1) and cullin 4 (Cul4) and subsequent proteasomal degradation has been found to be stimulated by binding to the human immunodeficiency virus (HIV) accessory protein viral protein R (Vpr) [27]. This is thought to be a mechanism for reducing the number of AP sites in viral reverse transcripts, as a consequence of UNG and SMUG1 activity on uracil residues generated by the cellular enzyme apolipoprotein B mRNA editing enzyme 3 (APOBEC3), and therefore enhancing viral replication. There is a body of evidence suggesting that modification of TDG with the small ubiquitin-like modifier (SUMO)-1, -2 and -3 at lysine 330, located in the C-terminal region may be important for its activity [28–31]. Indeed, a non-covalent interaction of SUMO with this C-terminal region of TDG reduces its AP site binding affinity [28] by altering the conformation of the protein, thereby competitively inhibiting its DNA-binding domain [30,31]. Given
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that both ubiquitylation and SUMOylation occur on lysine residues, it would be interesting to investigate whether the respective sites on TDG overlap or are distinct. Consequently, the two modifications may work in parallel whereby SUMO alters its AP site affinity to promote dissociation from the DNA after the damaged base has been excised, and the addition of ubiquitin would target TDG for proteasomal degradation.
End processors APE1 is the major AP endonuclease activity employed during BER. The first evidence for the existence of an E3 ubiquitin ligase regulating APE1 was the observation that following DNA damage, APE1 was polyubiquitylated on lysines 24, 25 and 27 by the mouse double minute 2 (MDM2) enzyme [32]. MDM2 is more commonly known to regulate protein levels of the p53 tumour suppressor protein that acts as a transcription factor mediating the cellular response to genotoxic stress. Indeed, APE1 polyubiquitylation was found to be dependent on the presence of p53. However, these data have been superseded by evidence that the major E3 ubiquitin ligase activity for APE1 is ubiquitin protein ligase E3 component n-recognin 3 (UBR3), and not MDM2 [33]. This was discovered by employing APE1 as a substrate in an in vitro ubiquitylation assay using fractionated HeLa cell extracts generated by column chromatography. However, similar to the first study, UBR3 polyubiquitylated APE1 within its N-terminus, and specifically this was spread over nine lysine residues within the first 35 amino acids on the protein. Key evidence for demonstrating that UBR3 is a key regulator of APE1 protein levels was provided by the fact that mouse embryonic fibroblasts (MEFs) lacking the Ubr3 gene not only have increased abundance of APE1 as a result of the lack of ubiquitylation-dependent degradation, but also that these cells showed signs of genetic instability as evidenced by increased DNA double strand break formation [33]. Consequently, it was suggested that an imbalance in the level of APE1, in comparison to the downstream enzyme Pol β, causes a build-up of DNA single strand breaks and that when cells progress through DNA replication, these lesions induce replication fork stalling leading to double strand break formation. It is interesting to note that acetylation has been observed on lysine residues in the same unstructured N-terminal region of APE1 which is ubiquitylated [34]. Acetylation of these residues alters the structure of this region and leads to binding of the deacetylase sirtuin 1, which causes deacetylation of APE1, and is thought to promote BER by increasing binding of the protein to XRCC1 and enhancing AP endonuclease activity [35]. As the N-terminal region has been proposed to mediate the interaction between APE1 and both rRNA and the nucleolar protein nucleophosmin, it would be interesting to investigate the effects of cross-talk between ubiquitylation and acetylation on the enzymatic activity and protein levels of APE1, and the consequences for the efficiency of BER and genome stability. BER initiated by the NEIL DNA glycosylases generates a single nucleotide gap containing a 30 -phosphate end which requires further processing by PNKP to allow gap filling and ligation to proceed (Fig. 1). Recently, an interplay between phosphorylation and ubiquitylation of PNKP has been uncovered which has been shown to regulate cellular PNKP protein levels in response to DNA damage. While PNKP phosphorylation on serines 114 and 126 had previously been shown to be mediated by the protein kinase,
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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ataxia telangiectasia mutated (ATM) and to regulate DNA double strand break repair [36,37], this site-specific PNKP phosphorylation was also observed in HCT116p53þ/þ cells in response to oxidative stress [38]. Concurrent with this ATM-dependent phosphorylation was an approximate 1.5-fold increase in PNKP protein levels peaking at 4–6 h post-treatment which correlated with a decrease in PNKP polyubiquitylation. The E3 ubiquitin ligase targeting PNKP for ubiquitylation was consequently purified from HeLa cell extracts and found to consist of a complex containing Cul4A, DNA damage binding protein 1 (DDB1) and a WD-40 repeat protein called serine–threonine kinase receptor-associated protein (STRAP). WD-40 repeat proteins, such as STRAP, are the adaptor proteins that interact with DDB1 and which provide specificity of the multiple Cul4A–DDB1 associated E3 ubiquitin ligase complexes to substrate proteins [39]. The sites of PNKP ubiquitylation by Cul4A–DDB1–STRAP were found to be lysines 414, 417 and 484 and Strap knockout MEFs demonstrated an increase in PNKP protein levels, and resistance to oxidative stress [38]. Furthermore, the link between ATM-dependent phosphorylation and inhibition of Cul4A–DDB1–STRAP dependent ubiquitylation was provided by evidence that a phosphomimetic S114/ 126E PNKP mutant displayed reduced ubiquitylation by Cul4A– DDB1–STRAP in vitro and in vivo, and consequently the protein was more stable when introduced into HCT116p53þ/þ cells. This suggests that phosphorylation of PNKP by ATM is directly responsible for the stabilisation of PNKP in response to oxidative stress, and provides a potential explanation for the increased sensitivity of ATM deficient cells to oxidative stress [40].
Gap fillers The stability of Pol β, the major DNA polymerase employed during BER, has been discovered to be dependent on the presence of XRCC1 on damaged DNA [41]. Consequently, using Pol β as a substrate in an in vitro ubiquitylation system combined with fractionated HeLa cell extracts generated by column chromatography, the major E3 ubiquitin ligase targeting Pol β for polyubiquitylation was shown to be the E3 ubiquitin ligase CHIP, and that the site was localised to the 8 kDa N-terminal domain of Pol β [41]. Indeed, overexpression of CHIP in HeLa cells decreased cellular Pol β protein levels, and conversely, small interfering RNA (siRNA) knockdown of CHIP increased protein levels. Subsequent to this study, another E3 ubiquitin ligase activity for Pol β was discovered, this time supporting monoubiquitylation [42]. Monoubiquitylation was found to occur on lysines 41, 61 and 81 by the E3 ubiquitin ligase Mule/ARF-BP1, which compartmentalised the protein to the cytoplasm. In fact, monoubiquitylation on these site-specific residues of Pol β was found to be a prerequisite for subsequent polyubiquitylation by CHIP, as shown by modulation in monoubiquitylated Pol β protein levels following CHIP overexpression or siRNA knockdown in HeLa cells. The E3 ubiquitin ligase activity of Mule/ARF-BP1 is known to be inhibited by the tumour suppressor protein ARF [43], and consequently siRNA knockdown of ARF in HeLa cells caused an increase in Pol β monoubiquitylation [42]. Regulation of Pol β protein levels by Mule and ARF was furthermore shown to alter the proficiency of HeLa cells to undergo DNA damage repair induced by oxidative stress. Since ubiquitylation of proteins is a reversible modification, it was further predicted that a DUB capable of recycling Pol β from the monoubiquitylated pool contained within the cytoplasm may
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exist, which would allow cellular Pol β protein levels to adjust to the DNA damage environment. Indeed, using ubiquitylated Pol β as a substrate in an in vitro deubiquitylation system combined with fractionated HeLa cell extracts generated by column chromatography, the ubiquitin-specific protease 47 (USP47) was purified as the major DUB activity for Pol β [44]. DUB activity was confirmed in vitro using recombinant USP47 expressed and purified from bacterial cells, which interestingly was able to deubiquitylate both monoubiquitylated Pol β (generated by Mule) and polyubiquitylated Pol β (generated by CHIP). It was further discovered that USP47 regulates the cellular levels of Pol β, since siRNA knockdown of USP47 in HeLa cells resulted in an increase in monoubiquitylated Pol β levels found in the cytoplasm, and as a consequence caused a reduction in the cytoplasmic levels of the native protein. Interestingly, a simultaneous siRNA knockdown of Mule and USP47 was discovered to be able to restore exogenously expressed Pol β levels that were reduced by a USP47 siRNA knockdown alone, demonstrating that Mule was specifically targeting newly synthesised Pol β for ubiquitylation-dependent degradation. As a consequence of USP47-dependent regulation of cellular Pol β levels, a delay in the rate of repair of hydrogen peroxide and methyl methanesulfonate (MMS)-induced DNA damage, as well as reduced cell survival following hydrogen peroxide treatment, was observed in HeLa cells lacking USP47. The dRP lyase activity of Pol β is known to be mediated by Schiffbase formation using lysine 72, which has also previously been shown to be negatively regulated by acetylation by p300 [45]. Given the close proximity of this residue to those monoubiquitylated by Mule on lysines 41, 61 and 81, there is potential for crosstalk between these two regulatory modifications despite these appearing to occur in separate compartments of the cell. Nevertheless, it would be interesting to examine this proposed crosstalk, particularly during the DNA damage response. These studies have collectively demonstrated an important role for CHIP, Mule and USP47 in the regulation of cellular Pol β protein levels, and consequently in the cellular response to DNA damage. However, this mechanism appears to be calibrated to allow small changes in Pol β protein levels, presumably as a consequence of minor fluctuations in endogenous DNA damage generated through cellular metabolism. Therefore any significant, sustained elevations in DNA damage through exogenous stress which exceed cellular DNA repair capacity are likely to drive the cell into apoptosis.
Nick sealers The scaffold protein XRCC1 is in a stable complex with Lig III and the importance of cellular XRCC1 protein levels is highlighted by the fact that XRCC1 deficient cells contain reduced levels of Lig III, but also Pol β, and display hypersensitivity to DNA damaging agents [41,47]. Both XRCC1 and Lig III have been shown to be polyubiquitylated in vitro by CHIP, the same E3 ubiquitin ligase responsible for polyubiquitylation of Pol β [41]. Furthermore, knockdown of CHIP in HeLa cells using siRNA increased the stability of both XRCC1 and Lig III, showing CHIP-dependent ubiquitylation results in their degradation by the UPP. The site of ubiquitylation of XRCC1 was localised to the C-terminal region of the protein containing a BRCA1 C-terminus (BRCT) motif, since a truncated form of the protein lacking this region showed increased stability in comparison to the wild-type protein when transfected into XRCC1-deficient cells [41].
Please cite this article as: M.J. Edmonds, J.L. Parsons, Regulation of base excision repair proteins by ubiquitylation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.07.031
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Both XRCC1 and Lig III are also subject to ubiquitylation by another E3 ubiquitin ligase activity which was identified as Iduna, or ring finger protein 146 (RNF146), although this is dependent on XRCC1 and Lig III being modified by poly(ADP-ribosyl)ation beforehand [46]. Iduna was found to protect against cell death induced by ionising radiation or treatment with the alkylating agent N-methylN-nitro-N-nitrosoguanidine (MNNG), although due to the number of targets identified for Iduna-dependent ubiquitylation, it is unclear whether this effect is mediated predominantly through XRCC1 and Lig III, and therefore requires further study.
Future perspectives The study of the regulation of components of the BER pathway, particularly through the UPP, is still at a very early stage and therefore the E3 ubiquitin ligases, the DUBs and consequently the mechanisms controlling BER proteins are still unclear. In particular, little is known about the regulation of the enzymes performing the initial step of BER, the DNA glycosylases, although there is evidence from proteomic screens that they are targets for ubiquitylation. It will be interesting to see in the future whether common themes emerge, for example, whether particular classes of BER enzymes are regulated by the same E3 ubiquitin ligases/ DUBs, or whether each BER component has its own specific complement of ubiquitylation/deubiquitylation enzyme(s). Interestingly, misregulation of BER protein levels has been observed in a range of human diseases, particularly in cancer but also in premature ageing and neurodegenerative diseases. Therefore, once uncovered, the next step would be to investigate the role of E3 ubiquitin ligases/DUBs targeting BER proteins, for their involvement in BER misregulation found in cells and tissues derived from patients with these diseases. Consequently, this detailed knowledge may help to uncover novel targets for drugs or small molecule inhibitors, which when combined with radiotherapy and/or chemotherapy could enhance current treatment regimens for patients with diseases containing altered BER protein expression.
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Acknowledgement
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Jason Parsons is supported by funding from North West Cancer Research Fund (CR972 and CR1016) and by the Medical Research Council via a New Investigator Research Grant (MR/M000354/1).
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