Abstract

DNA suffers constant insult from a variety of endogenous and exogenous sources. To deal with the arising lesions, cells have evolved complex and coordinated pathways, collectively termed the DNA damage response (DDR). Importantly, an improper DDR can lead to genome instability, premature ageing and human diseases, including cancer as well as neurodegenerative disorders. As a crucial process for cell survival, regulation of the DDR is multi-layered and includes several post-translational modifications. Since the discovery of ubiquitin in 1975 and the ubiquitylation cascade in the early 1980s, a number of ubiquitin-like proteins (UBLs) have been identified as post-translational modifiers. However, while the importance of ubiquitin and the UBLs SUMO and NEDD8 in DNA damage repair and signalling is well established, the roles of the remaining UBLs in the DDR are only starting to be uncovered. Herein, we revise the current status of the UBLs ISG15, UBL5, FAT10 and UFM1 as emerging co-regulators of DDR processes. In fact, it is becoming clear that these post-translational modifiers play important pleiotropic roles in DNA damage and/or associated stress-related cellular responses. Expanding our understanding of the molecular mechanisms underlying these emerging UBL functions will be fundamental for enhancing our knowledge of the DDR and potentially provide new therapeutic strategies for various human diseases including cancer.

Introduction

The genome is constantly subjected to injuries arising from endogenous and exogenous factors, such as ultraviolet (UV) light, ionizing radiation (IR), carcinogens and reactive radicals, which create different types of DNA lesions that pose harm to cell viability. To respond to such insults, cells have evolved elaborate and coordinated pathways, collectively termed the DNA damage response (DDR). The DDR senses and signals different types of DNA damage, halts cell cycle progression to initiate repair pathways and promotes senescence or apoptosis if the damage is too severe [1,2]. Given that chromatin is the substrate for DNA repair, the DDR also regulates chromatin structure, for example, to facilitate accessibility of DNA repair factors and to co-ordinate repair with other ongoing chromatin processes such as transcription [3]. The DDR is orchestrated by kinases belonging to the phosphoinositide 3-kinase-like kinase (PIKK) family, namely ATM, ATR and DNA-PKcs. These kinases phosphorylate and thereby activate DNA repair and signalling factors as well as other downstream effectors to amplify the DDR. One example is p53, which is activated by the DDR to halt the cell cycle in G1 and induce senescence or apoptosis (Figure 1) [4]. Importantly, failure to properly detect and/or repair damaged DNA can lead to physical blocks in the replication and transcription machinery. In addition, mutations and chromosomal aberrations can arise from misrepair and lead to genome instability, a hallmark of cancer [1,5]. Furthermore, hereditary DDR defects are associated with predisposition to immunodeficiency, neurodegeneration, infertility, premature ageing and most notably tumorigenesis [1,5,6], highlighting the importance of DDR pathways to human health.

Overview of key DNA damage response aspects regulated by ISG15, FAT10, UBL5 and UFM1

Figure 1
Overview of key DNA damage response aspects regulated by ISG15, FAT10, UBL5 and UFM1

Following DNA damage, DNA lesions are detected by specific DNA damage response (DDR) sensors, which recruit and activate the PIKKs ATM, ATR and/or DNA-PKcs responsible for phosphorylating downstream DDR factors such as p53 to induce cell cycle arrest, senescence or apoptosis. Note that several UBLs (ISG15, FAT10, UBL5 and UFM1) have emerged as DDR co-regulators, and can help activate the DDR (e.g. UFM1 promotion of ATM activation, red arrow) and/or impact on downstream DDR events. The DDR includes repair by multiple pathways depending on type of DNA lesion, for example, stalled replication forks, DNA interstrand cross-links (ICLs) or DNA double-strand breaks (DSBs). To date, ISGylation of p53 and PCNA, FATylation of p53 and PCNA, interaction of FANCI with UBL5, and UFMylation of MRE11 (part of the MRN complex) and histone H4 have been linked to different aspects of the DDR as indicated; Abbreviation: PIKK, phosphoinositide 3-kinase-like kinase.

Figure 1
Overview of key DNA damage response aspects regulated by ISG15, FAT10, UBL5 and UFM1

Following DNA damage, DNA lesions are detected by specific DNA damage response (DDR) sensors, which recruit and activate the PIKKs ATM, ATR and/or DNA-PKcs responsible for phosphorylating downstream DDR factors such as p53 to induce cell cycle arrest, senescence or apoptosis. Note that several UBLs (ISG15, FAT10, UBL5 and UFM1) have emerged as DDR co-regulators, and can help activate the DDR (e.g. UFM1 promotion of ATM activation, red arrow) and/or impact on downstream DDR events. The DDR includes repair by multiple pathways depending on type of DNA lesion, for example, stalled replication forks, DNA interstrand cross-links (ICLs) or DNA double-strand breaks (DSBs). To date, ISGylation of p53 and PCNA, FATylation of p53 and PCNA, interaction of FANCI with UBL5, and UFMylation of MRE11 (part of the MRN complex) and histone H4 have been linked to different aspects of the DDR as indicated; Abbreviation: PIKK, phosphoinositide 3-kinase-like kinase.

Different DNA repair or bypass pathways are dedicated to distinct types of lesions [1]. A DNA damage tolerance mechanism called translesion DNA synthesis (TLS) allows cells to replicate past DNA lesions that would otherwise block the replication machinery (Figure 1) [1,2]. DNA interstrand cross-links (ICLs) that can be caused by a variety of exogenous or endogenous agents are repaired by the Fanconi anemia (FA) pathway (Figure 1) [1]. Moreover, DNA double-strand breaks (DSBs) induced for instance by IR are mainly repaired by one of two major pathways: homologous recombination (HR) or non-homologous end-joining (NHEJ). The former is a high-fidelity process restricted to G2/S cell cycle phases that takes advantage of the sister chromatid as a homologous template for repair (Figure 1). The latter is a process that can be error-prone, but allows for repair throughout the cell cycle, except for mitosis [1,2,7]. Other repair pathways include, but are not limited to, base excision repair (BER) to repair non-helix distorting base damage, nucleotide excision repair (NER) to repair helix-distorting damage and mismatch repair (MMR) to fix base mismatches [1,2].

Strikingly, the human cellular proteome consists of more than a million distinct proteins/protein isoforms, while our genome encodes only ∼20,000 genes. The exponential expansion of the human proteome is substantially based on protein post-translational modifications (PTMs) and to a lesser extent, alternative promoter usage, as well as splicing, to generate different protein isoforms. In fact, PTMs are an essential regulatory component for virtually all cellular activities including the DDR (Figure 1) [8]. This is exemplified by ubiquitylation, one of the most studied PTMs, which involves conjugation of ubiquitin, a highly conserved 8.5 kD, 76 amino-acid protein, onto mostly lysines of target proteins. Ubiquitylation proceeds via a three-step ATP-dependent enzymatic cascade formed by an E1-activating, E2-conjugating and E3-ligating enzyme. In some instances, the ubiquitylation cascade can be complemented by E4 factors that enhance E3-mediated ubiquitin transfer to substrates [9–11]. Following the discovery of ubiquitin, more than a dozen ubiquitin-like proteins (UBLs) have been identified, including several paralogues of small ubiquitin-like modifier (SUMO), neural precursor cell expressed and developmentally down-regulated 8 (NEDD8), interferon-stimulated gene 15 (ISG15), human leukocyte antigen F locus adjacent transcription 10 (FAT10), ubiquitin-fold modifier 1 (UFM1), ubiquitin-related modifier 1 (URM1), autophagy-related protein 12 (ATG12), autophagy-related protein 8 (ATG8), Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FUBI) and ubiquitin-like protein 5 (UBL5) [11]. Although UBLs differ considerably in their primary amino-acid sequence, they all share a similar 3D structure to ubiquitin, including a β-grasp fold and commonly a di-glycine-containing C-terminal tail for covalent attachment to substrate proteins via the E1-E2-E3 enzymatic cascade (Figure 2) [11,12]. While the involvement of ubiquitin, SUMO and NEDD8 in the DDR has been well established, the roles of the remaining UBLs in this regard have only started to emerge (Figure 1). Herein, we review recent findings that implicate the next generation of UBLs in the DDR, specifically ISG15, FAT10, UBL5 and UFM1, highlighting future perspectives for this novel and exciting research field.

Key features of UBL systems (ISG15, FAT10, UBL5 and UFM1) in comparison with ubiquitin

Figure 2
Key features of UBL systems (ISG15, FAT10, UBL5 and UFM1) in comparison with ubiquitin

(A) Top panel - 3D structures of ubiquitin, ISG15 N- and C-terminal UBL domains (represented in a single structure), FAT10 N- and C-terminal UBL domains (depicted as separate moieties), UBL5 and UFM1. UBL structures (slate blue) are overlaid with ubiquitin (grey) for comparison; ISG15 and FAT10 N- and C-terminal UBL domains represented as N and C, respectively; FAT10 N-terminal UBL structure is incomplete (residues 10–86). Key C-terminal di-glycines (GGs), or mono-glycine (G) in case of UFM1, required for conjugation are highlighted in red for UBLs and green for ubiquitin. The single C-terminal glycine of UBL5 is insufficient for conjugation and hence, UBL5 is considered a non-covalent modifier; ISG15 C-terminal di-glycine has been included manually due to its absence in the corresponding PDB file. Bottom panel: electrostatic potentials of ubiquitin and UBL surfaces (longest Ub/UBL axis aligned to x-axis). PDB files are as follows: ubiquitin (2LJ5, NMR structure), ISG15 (3RT3, X-ray crystal structure), N-terminal FAT10 UBL (6GF1, X-ray crystal structure), C-terminal FAT10 UBL (6GF2, NMR structure), UBL5 (1P0R, NMR structure) and UFM1 (1WXS, NMR structure). (B) Schematic illustrating key features of UBL systems in comparison with ubiquitin. Numbers/names for enzymatic components of ubiquitin system are according to [47,108,112], as referenced in the Figure; for UBL system, only enzymes with known cellular activity are included. As a non-covalent modifier, UBL5 is depicted separately. Ub: ubiquitin; UBL: ubiquitin-like modifier; N: N-terminal; C: C-terminal; NMR: nuclear magnetic resonance. $For further information on validated DNA damage response targets of ISG15, FAT10, UBL5 and UFM1 see Table 1. *ISG15 not present in yeast, nematode (Caenorhabditis elegans) or fly (Drosophila sp). **Deubiquitylase/deUBLylase-mediated processing/maturation step is typically required to expose C-terminal residue(s) for ubiquitin/UBL conjugation to target residues. ***Information based on ‘The Human Protein Atlas’ [113]. Note that several UBLs are specifically induced in response to certain stimuli (see Table 1 and main text for further information).

Figure 2
Key features of UBL systems (ISG15, FAT10, UBL5 and UFM1) in comparison with ubiquitin

(A) Top panel - 3D structures of ubiquitin, ISG15 N- and C-terminal UBL domains (represented in a single structure), FAT10 N- and C-terminal UBL domains (depicted as separate moieties), UBL5 and UFM1. UBL structures (slate blue) are overlaid with ubiquitin (grey) for comparison; ISG15 and FAT10 N- and C-terminal UBL domains represented as N and C, respectively; FAT10 N-terminal UBL structure is incomplete (residues 10–86). Key C-terminal di-glycines (GGs), or mono-glycine (G) in case of UFM1, required for conjugation are highlighted in red for UBLs and green for ubiquitin. The single C-terminal glycine of UBL5 is insufficient for conjugation and hence, UBL5 is considered a non-covalent modifier; ISG15 C-terminal di-glycine has been included manually due to its absence in the corresponding PDB file. Bottom panel: electrostatic potentials of ubiquitin and UBL surfaces (longest Ub/UBL axis aligned to x-axis). PDB files are as follows: ubiquitin (2LJ5, NMR structure), ISG15 (3RT3, X-ray crystal structure), N-terminal FAT10 UBL (6GF1, X-ray crystal structure), C-terminal FAT10 UBL (6GF2, NMR structure), UBL5 (1P0R, NMR structure) and UFM1 (1WXS, NMR structure). (B) Schematic illustrating key features of UBL systems in comparison with ubiquitin. Numbers/names for enzymatic components of ubiquitin system are according to [47,108,112], as referenced in the Figure; for UBL system, only enzymes with known cellular activity are included. As a non-covalent modifier, UBL5 is depicted separately. Ub: ubiquitin; UBL: ubiquitin-like modifier; N: N-terminal; C: C-terminal; NMR: nuclear magnetic resonance. $For further information on validated DNA damage response targets of ISG15, FAT10, UBL5 and UFM1 see Table 1. *ISG15 not present in yeast, nematode (Caenorhabditis elegans) or fly (Drosophila sp). **Deubiquitylase/deUBLylase-mediated processing/maturation step is typically required to expose C-terminal residue(s) for ubiquitin/UBL conjugation to target residues. ***Information based on ‘The Human Protein Atlas’ [113]. Note that several UBLs are specifically induced in response to certain stimuli (see Table 1 and main text for further information).

ISG15: more than a protective layer against viruses

ISG15 was the first UBL to be discovered and contains two UBL domains (Figure 2A), each with ∼30% amino-acid sequence homology to ubiquitin (Figure 2B), that are joined by a flexible linker [13–17]. ISG15 encodes a 165 amino-acid inactive precursor (17 kD) from which 8 C-terminal amino acids are cleaved off by an enzyme called USP18 (also known as UBP43), to expose a di-glycine in the mature form of ISG15 (15 kD) used for substrate conjugation (Figure 2) [18]. Like ubiquitylation, ISGylation occurs through a three-step enzymatic cascade starting with an E1, UBA7 (also known as UbE1L) [19–22], followed by one known E2 that is functional in cells called UBE2L6 (also known as UBCH8) [19,22]. One of three known E3 ligases, HERC5, HHARI and EFP (also known as TRIM25), then facilitates ISG15 conjugation to lysines of target proteins (Table 1) [23–26]. Of note, while EFP and HHARI exhibit some substrate specificity, HERC5 promiscuously ISGylates newly translated proteins at polyribosomes [27]. ISG15 can also be conjugated to K29 of ubiquitin to form mixed ISG15-ubiquitin chains [28]. Finally, USP18, the same enzyme that processes the ISG15 precursor, deconjugates ISG15 from proteins, making ISGylation a reversible process (Figure 2B) [29,30]. Importantly, the expression of the majority of ISGylation factors is stimulated by type I interferons, and consistent with this inducibility, ISG15 is largely known for its role in the classical immune response during viral infections [31].

Table 1
Selected putative/validated human UBL targets relevant for the DNA damage response
UBLStimuli$Cellular pathwaysTargets relevant for DNA damage response#
ISG15 Interferon-type I signalling [31], HU, MMS [32], UV light [32,35,40], Dox [35] and CPT [35,41Cell cycle signalling and apoptosis
Chromatin remodelling
DSB repair / ICL
NER / BER
TLS
Others 
CBX4 [37], p53* [33–35], ∆Np63α (p63)* [45]
CHD1, RBBP4, SAFA [37]
UBE2N* [46], XRCC5, XRCC6 [37]
DDB2 [37], XPD* [39]
PCNA* [32],
VCP* [37,38
FAT10 IFNγ, TNFα, UV light, IR and etoposide (VP16) [77Cell cycle signalling and apoptosis
Chromatin remodelling
DSB repair / ICL
NER / BER /SSA
TLS
Others 
CBX4, HAT1, p53* [70]
HDAC3, HDAC6, KDM1A, RBBP7 [70], H2AX, RBBP4, KAP1 [71]
DNA-PKcs [69,71], BRCC45, FANCI, FANCJ, FANCL, XRCC6 [70]
DBB1, MSH3, PARP1 [69], MSH6 [69,71], MSH2 [70]
PCNA* [70,71]
MCM2 [69], MCM4, p62* [70], MCM3, VCP [71
UBL5 ND Chromatin remodelling

DSB repair / ICL
MMR / NER / BER
TLS
Others 
CHD4, KAP1, HDAC1, HDAC6, HP1α, HP1β, HP1γ, SAFA, SAFB, RBBP4, 53BP1 [88]
BCCIP, BRCC3, BRCC45, DNA-PKcs, FANCI*, MERIT40, MRE11, NBS1, RAD50, RAD54L, RPA70, RIF1, XRCC5, XRCC6 [88]
DDB1, FEN1, LIG1, LIG3, MSH2, PMS2, MLH1, MSH6, RAD21, RECQ1, XDP, XRN2 [88]
PCNA [88]
MCM2, MCM4, MCM5, MCM6, MCM7, VCP [88
UFM1 ER stress and UPR [92,98,100Cell cycle signalling and apoptosis
Chromatin remodelling
DSB repair / ICL
BER / SSA
TLS
Others 
CBX4 [102]
CHD3, CHD4 [101], KAP1, RBBP4, RBBP7 [101,102], CHD1, CHD8, HAT1, HDAC1, RNF111, SAFB, SAFB2 [102]
DNA-PK, MDC1 [101], MRE11*, H4*, XRCC5, XRCC6 [101,102], APTX, BRCC3, UBE2N [102]
PARP1 [101]
PCNA [101,102]
MCM3, VCP [101], MCM2 [102
UBLStimuli$Cellular pathwaysTargets relevant for DNA damage response#
ISG15 Interferon-type I signalling [31], HU, MMS [32], UV light [32,35,40], Dox [35] and CPT [35,41Cell cycle signalling and apoptosis
Chromatin remodelling
DSB repair / ICL
NER / BER
TLS
Others 
CBX4 [37], p53* [33–35], ∆Np63α (p63)* [45]
CHD1, RBBP4, SAFA [37]
UBE2N* [46], XRCC5, XRCC6 [37]
DDB2 [37], XPD* [39]
PCNA* [32],
VCP* [37,38
FAT10 IFNγ, TNFα, UV light, IR and etoposide (VP16) [77Cell cycle signalling and apoptosis
Chromatin remodelling
DSB repair / ICL
NER / BER /SSA
TLS
Others 
CBX4, HAT1, p53* [70]
HDAC3, HDAC6, KDM1A, RBBP7 [70], H2AX, RBBP4, KAP1 [71]
DNA-PKcs [69,71], BRCC45, FANCI, FANCJ, FANCL, XRCC6 [70]
DBB1, MSH3, PARP1 [69], MSH6 [69,71], MSH2 [70]
PCNA* [70,71]
MCM2 [69], MCM4, p62* [70], MCM3, VCP [71
UBL5 ND Chromatin remodelling

DSB repair / ICL
MMR / NER / BER
TLS
Others 
CHD4, KAP1, HDAC1, HDAC6, HP1α, HP1β, HP1γ, SAFA, SAFB, RBBP4, 53BP1 [88]
BCCIP, BRCC3, BRCC45, DNA-PKcs, FANCI*, MERIT40, MRE11, NBS1, RAD50, RAD54L, RPA70, RIF1, XRCC5, XRCC6 [88]
DDB1, FEN1, LIG1, LIG3, MSH2, PMS2, MLH1, MSH6, RAD21, RECQ1, XDP, XRN2 [88]
PCNA [88]
MCM2, MCM4, MCM5, MCM6, MCM7, VCP [88
UFM1 ER stress and UPR [92,98,100Cell cycle signalling and apoptosis
Chromatin remodelling
DSB repair / ICL
BER / SSA
TLS
Others 
CBX4 [102]
CHD3, CHD4 [101], KAP1, RBBP4, RBBP7 [101,102], CHD1, CHD8, HAT1, HDAC1, RNF111, SAFB, SAFB2 [102]
DNA-PK, MDC1 [101], MRE11*, H4*, XRCC5, XRCC6 [101,102], APTX, BRCC3, UBE2N [102]
PARP1 [101]
PCNA [101,102]
MCM3, VCP [101], MCM2 [102

*Experimentally validated targets.

$

Stressors known to induce expression of respective UBLs.

#

Target lysines (residues required for non-covalent interaction in case of UBL5) and locations within target proteins. For ISGylation: p53 (multiple sites in N- and C-terminal transactivation and tetramerization domains, including K291 and K292 located close to p53 DNA-binding domain), ∆Np63α (K139 and K324 in p53 DNA-binding domain), UBE2N (K92 near catalytic cysteine C87), PCNA (K164 and K168 in C-terminal domain), target residues for XPD and VCP ISGylations were not investigated; for FATylation: p53- and PCNA-specific residues of FAT10 conjugation were not experimentally established; for UBL5 interaction: FANCI (E268 and H274 located in Solenoid 1 domain); for UFMylation: MRE11 (K281/K282 close to MRE11 DNA-binding domain), Histone H4 (K31 in centromere kinetochore component CENP-T histone fold).

Abbreviations: BER, base excision repair; Dox, doxorubicin; DSB, DNA double-strand break; ER, endoplasmic reticulum; HU, hydroxyurea; ICL, interstrand cross-link; IFN, interferon; MMR, mismatch repair; MMS, methyl methane sulfonate; ND, not determined; NER, nucleotide excision repair; SSA, single-strand annealing; TLS, translesion synthesis; TNF, tumor necrosis factor; UBL, ubiquitin-like modifier; UPR, unfolded protein response.

A pleiotropic p53 modulator

In addition to the above, ISGylation has recently been implicated in downstream DDR events [32–35], consistent with the proteomic identification of several DDR factors as putative ISG15-interacting proteins (Table 1) [36–39]. Indeed, various ISGylation factors contain p53-responsive elements in their promoters [35], and consequently, ISGylation can be stimulated by genotoxic stress, such as UV irradiation, doxorubicin, and camptothecin, which are all known to induce p53 expression [35,40–42]. Notably, DNA damage can induce ISGylation of a different set of substrates compared with interferons [35,42], including p53 itself, which is targeted by ISG15 at several lysines including K291 and K292 [33–36]. However, the effects of ISG15 on wild-type and mutant p53 are complex (Figure 3A,B). In fact, ISGylation has been reported to promote p53 transactivity by increasing its phosphorylation and acetylation statuses, which, in turn, enhances its binding to target promoter genes. The resulting positive feedback loop reinforces the tumour-suppressive functions of p53 under genotoxic stress (Figure 3B) [35]. Together with polyubiquitylation, ISG15 is also involved in p53 stability and cisplatin resistance [33,34,43,44]. In healthy cells, ISG15 is preferentially conjugated to misfolded and dominant-negative p53. These events lead to degradation of misfolded p53 by the proteasome, which promotes wild-type p53 function (Figure 3A, left) [33]. By contrast, in certain cancer contexts and dependent on oncogene activity, p53 ISGylation can become unspecific and also target wild-type p53 for degradation, thereby causing an overall decrease in p53 activity (Figure 3A, right) [34]. Different E3 ligases have been reported to ISGylate p53 including EFP and HERC5 [33–35]. In addition, ISGylation of other p53 family members, such as p63, has been observed and linked to abnormal cellular growth and tumorigenesis (Figure 3C) [45]. Overall, it appears that the cellular responses and precise location of ISGylations on wild-type and mutant p53, as well as the contributing ISGylation factors, depend on the cellular context and extracellular stimuli. Collectively, these findings highlight ISGylation as a regulatory layer that helps cells to fine-tune and adapt downstream DDR responses mediated by p53.

DDR pathways regulated by ISG15

Figure 3
DDR pathways regulated by ISG15

(A) HERC5-mediated p53 ISGylation. In healthy cells (left), HERC5-mediated ISGylation of p53 primarily removes misfolded p53 via proteasomal degradation, thereby increasing wild-type (WT) p53 activity. In cancer cells (right), oncogene proteins, such as SRC, RAS and MYC, enhance the interaction between HERC5 and ISG15, leading to indiscriminate modification of native and misfolded p53 and an overall reduction of total anti-tumoural p53 activity. (B) EFP-mediated p53 ISGylation. Upon DNA damage, p53 is phosphorylated (orange, P) and acetylated (yellow, Ac) by CHK1 and p300, respectively, resulting in dissociation of p53 from MDM2 and subsequent p53 stabilization. EFP conjugates ISG15 to stabilized p53 at lysines K291 and K292, which increases phosphorylation and acetylation of p53, as well as its ability to bind p53 responsive elements (p53REs), thereby inducing expression of p53 targets, such as p21, BAX, CDKN1, p53 itself and ISGylation factors. Increased expression of ISGylation factors accelerates p53 ISGylation and transactivation, leading to tumour growth suppression. This positive feedback loop is switched off by USP18 via deISGylation of p53, leading to p53 destabilization. (C) Ablation of oncogenic functions of ∆Np63α, an isoform of the p53 family member p63, by ISG15 modification. DNA damage induces ISGylation of ∆Np63α at lysines K139 and K324, leading to CASP2-mediated cleavage and cytoplasmic release of the ∆Np63α C-terminus, termed transactivation inhibitor (TI). Cleaved ∆Np63α can no longer inhibit the transcriptional activities of other p53 family members, such as p53, TAp63α and TAp63γ, thus facilitating their anti-tumoural and apoptotic functions. (D) Model for termination of translesion DNA synthesis (TLS) by ISGylation of PCNA. Under non-stressed conditions, PCNA serves as a processivity factor for replicative DNA synthesis. In response to certain types of DNA damage induced, for example, by UV light, PCNA is monoubiquitylated at K164 in one of its three identical subunits by the RAD6/RAD18 E3 ligase complex, which recruits the translesion polymerase Polη to carry out TLS. After bypass of the lesion, EFP ISGylates PCNA at K168 possibly in a different subunit, triggering recruitment of USP10, which deubiquitylates PCNA and releases Polη to avoid UV-induced mutagenesis. ISGylation of PCNA at K164 probably prevents subsequent cycles of monoubiquitylation. Finally, USP18 deISGylates PCNA and allows reloading of replicative DNA polymerases as well as resumption of normal cell replication; Abbreviations: DDR, DNA damage response; POL, RNA polymerase II; Ub, ubiquitin.

Figure 3
DDR pathways regulated by ISG15

(A) HERC5-mediated p53 ISGylation. In healthy cells (left), HERC5-mediated ISGylation of p53 primarily removes misfolded p53 via proteasomal degradation, thereby increasing wild-type (WT) p53 activity. In cancer cells (right), oncogene proteins, such as SRC, RAS and MYC, enhance the interaction between HERC5 and ISG15, leading to indiscriminate modification of native and misfolded p53 and an overall reduction of total anti-tumoural p53 activity. (B) EFP-mediated p53 ISGylation. Upon DNA damage, p53 is phosphorylated (orange, P) and acetylated (yellow, Ac) by CHK1 and p300, respectively, resulting in dissociation of p53 from MDM2 and subsequent p53 stabilization. EFP conjugates ISG15 to stabilized p53 at lysines K291 and K292, which increases phosphorylation and acetylation of p53, as well as its ability to bind p53 responsive elements (p53REs), thereby inducing expression of p53 targets, such as p21, BAX, CDKN1, p53 itself and ISGylation factors. Increased expression of ISGylation factors accelerates p53 ISGylation and transactivation, leading to tumour growth suppression. This positive feedback loop is switched off by USP18 via deISGylation of p53, leading to p53 destabilization. (C) Ablation of oncogenic functions of ∆Np63α, an isoform of the p53 family member p63, by ISG15 modification. DNA damage induces ISGylation of ∆Np63α at lysines K139 and K324, leading to CASP2-mediated cleavage and cytoplasmic release of the ∆Np63α C-terminus, termed transactivation inhibitor (TI). Cleaved ∆Np63α can no longer inhibit the transcriptional activities of other p53 family members, such as p53, TAp63α and TAp63γ, thus facilitating their anti-tumoural and apoptotic functions. (D) Model for termination of translesion DNA synthesis (TLS) by ISGylation of PCNA. Under non-stressed conditions, PCNA serves as a processivity factor for replicative DNA synthesis. In response to certain types of DNA damage induced, for example, by UV light, PCNA is monoubiquitylated at K164 in one of its three identical subunits by the RAD6/RAD18 E3 ligase complex, which recruits the translesion polymerase Polη to carry out TLS. After bypass of the lesion, EFP ISGylates PCNA at K168 possibly in a different subunit, triggering recruitment of USP10, which deubiquitylates PCNA and releases Polη to avoid UV-induced mutagenesis. ISGylation of PCNA at K164 probably prevents subsequent cycles of monoubiquitylation. Finally, USP18 deISGylates PCNA and allows reloading of replicative DNA polymerases as well as resumption of normal cell replication; Abbreviations: DDR, DNA damage response; POL, RNA polymerase II; Ub, ubiquitin.

A direct role in translesion synthesis (TLS)

Recently, ISG15 was discovered to play a direct role in TLS to facilitate replication past DNA lesions induced by UV light [32]. A key event for initiating TLS is the monoubiquitylation of the proliferating cell nuclear antigen (PCNA) by RAD6/RAD18 at K164 [9], which promotes the displacement of the replicating polymerases, POLδ or Polɛ, with lower-fidelity TLS polymerases, POLη or POLζ. As a result, POLη and POLζ are capable of bypassing bulky DNA lesions, after which high-fidelity replication needs to be restored. To achieve this, the ISG15-E3 ligase EFP is tethered to monoubiquitylated PCNA where it generates monoISGylated PCNA at K168. ISGylated PCNA then recruits the deubiquitylase USP10, which removes ubiquitin from PCNA, thereby causing the release of Polη and other factors important for recruiting TLS polymerases, such as REV1, from PCNA, to terminate TLS. These events are followed by EFP-mediated conjugation of an additional ISG15 to K164 of PCNA, which probably prevents subsequent rounds of PCNA monoubiquitylation and TLS initiation. To finalize TLS, USP18 cleaves off ISG15 from PCNA for reloading of replicative DNA polymerases and resumption of DNA replication (Figure 3D) [32]. In line with these findings, ISGylation is crucial for preventing UV-mediated excessive mutagenesis and genome instability [32,42].

DDR modulation via cross-talk with ubiquitin

In addition to its direct function in terminating TLS, ISG15 may also regulate DSB repair by modifying the ubiquitin-E2 enzyme UBE2N (also known as UBC13) at K92 [46]. UBE2N is a well-established E2 important for a variety of DDR signalling events by generating K63-linked ubiquitin chains as docking platforms for DSB repair factors [9,47]. Interestingly, ISGylation inhibits UBE2N activity by disrupting its ability to form thioester bonds with ubiquitin [46]. However, the exact role this has in DNA damage repair remains to be determined, as well as any additional ISG15 functions in the DDR. Given the progress that has recently been made regarding the intersection of the immune response with the DDR, it will be interesting to see if, and to what extent, ISG15’s DDR functions intertwine with its immune response roles [31].

FAT10: early days for DDR regulation

FAT10 (18 kD) was discovered in 1996 and is exclusively found in mammals [48,49]. Like ISG15, it contains two ubiquitin-like moieties in tandem, which share ∼29% and ∼36% amino-acid identity to ubiquitin, respectively, and only ∼18% with each other (Figure 2A,B) [49,50]. Both FAT10 moieties display different surface charge distribution to ubiquitin and other UBLs, which explains their unique binding specificities (Figure 2A) [50]. FAT10 is expressed with a free C-terminal di-glycine directly available for activation and conjugation, thereby circumventing the processing step normally required for ubiquitin/UBL maturation (Figure 2B) [51]. FATylation starts with an E1, UBA6, followed by one E2 described to date, UBE2Z (also known as USE1) (Table 1) [52,53]. Both UBA6 and UBE2Z can also activate and conjugate ubiquitin, albeit with different kinetics [53–57]. Notably, neither E3s nor deFATylation enzymes have been discovered to date (Figure 2B). Therefore, it remains to be determined whether FAT10 requires E3 ligases for conjugation and if the modification is reversible. FAT10 is involved in the immune response, consistent with its restricted expression to immune system tissues under non-stressed conditions [58–60]. However, FAT10 is also ubiquitously induced by inflammatory cytokines, such as IFNγ and TNFα [60,61], retinoids [58], and viral infections [48,62–64]. Moreover, FAT10 expression is cell cycle-regulated [65]. Mechanistically, FAT10 has primarily been described as a signal for proteasomal degradation [66–69]. However, several studies suggest a wider range of FAT10 functions, including roles in the DDR (Table 1) [69–72], and mitotic regulation via non-covalent interaction between FAT10 and the spindle assembly checkpoint protein MAD2 [73–76].

p53 and PCNA – hubs for UBL regulation

In addition to the above, FAT10 expression is induced by DNA-damaging treatments, such as UV light, IR and etoposide [77]. These findings suggest functions for FATylation in the DDR, and indeed, a role for FAT10 in p53 transcriptional activation has been proposed, consistent with p53 being a substrate for FATylation (Figure 4A) [78]. However, similarly to ISG15, the effects are complex: on the one hand, overexpression of FAT10 leads to increased p53 transcriptional activity and altered p53 conformation and nuclear localization [78], while on the other hand, p53 was reported to down-regulate the expression of FAT10 [79]. Consistent with PCNA co-localizing with FAT10 in nuclear foci, PCNA has been validated as a FAT10 substrate [77], suggesting a role for FATylation in the DDR. Indeed, FATylation appears to regulate the cytoplasmic and nuclear stability of PCNA through the proteasome (Figure 4B). These discoveries suggest that FAT10 may contribute to the DDR by regulating PCNA turnover in a FATylation-dependent manner [77]. More studies are required to shed light on the exact mechanistic interplay between FAT10 and the DDR.

Model for DNA damage response regulation by FAT10

Figure 4
Model for DNA damage response regulation by FAT10

DNA damage caused by etoposide, UV light or ionizing radiation can up-regulate FAT10 expression. Overexpression of FAT10 leads to p53 FATylation (A), which increases its transcriptional activity (1) and reduces its accumulation in promyelocytic leukaemia nuclear bodies (PML-NBs, green dots) (2), suggestive of a role for FAT10 in promoting p53-mediated cellular DNA damage response pathways. Down-regulation of FAT10 expression by p53 suggests regulation of this pathway through a negative feedback loop. DNA damage-induced up-regulation of FAT10 expression also leads to PCNA FATylation (B), which increases in proportion to severity of DNA damage and is associated with proteasomal degradation. Some of the links above are extrapolated from separate studies; Abbreviation: POL, RNA polymerase II.

Figure 4
Model for DNA damage response regulation by FAT10

DNA damage caused by etoposide, UV light or ionizing radiation can up-regulate FAT10 expression. Overexpression of FAT10 leads to p53 FATylation (A), which increases its transcriptional activity (1) and reduces its accumulation in promyelocytic leukaemia nuclear bodies (PML-NBs, green dots) (2), suggestive of a role for FAT10 in promoting p53-mediated cellular DNA damage response pathways. Down-regulation of FAT10 expression by p53 suggests regulation of this pathway through a negative feedback loop. DNA damage-induced up-regulation of FAT10 expression also leads to PCNA FATylation (B), which increases in proportion to severity of DNA damage and is associated with proteasomal degradation. Some of the links above are extrapolated from separate studies; Abbreviation: POL, RNA polymerase II.

UBL5: sp(l)icing up genome stability as a non-covalent modulator

UBL5 (8.5 kD) is a 73 amino-acid protein that shares ∼22% amino-acid sequence identity with ubiquitin and displays a single C-terminal glycine that is insufficient for covalent conjugation to target proteins (Figure 2) [80]. UBL5 therefore represents an atypical UBL, which acts as a protein modulator through the formation of tight, non-covalent interactions with target proteins [81–82]. The wide expression and evolutionary conservation of UBL5 across eukaryotes including yeast, where it is known as Hub1, imply fundamental cellular functions [80,83,84]. In agreement with UBL5 localizing to Cajal bodies – nuclear domains important for assembly of spliceosomal components [85] – yeast studies demonstrated that UBL5 is important for pre-mRNA splicing [86,87]. Consistent with these findings, UBL5 associates with the pre-mRNA splicing machinery also in humans and is important for correct splicing of Sororin, a sister chromatid cohesion protector, important for maintaining genome stability [88]. A similar splicing role for UBL5 appears to apply to XRCC3, a protein important for DSB repair by HR, which is down-regulated when UBL5 is depleted (Figure 5) [88]. Taken together, these discoveries suggest that UBL5 function is important for indirectly promoting DNA repair and associated genome stability processes at the level of pre-mRNA splicing.

UBL5 promotes DNA damage response and associated pathways via non-covalent interactions

Figure 5
UBL5 promotes DNA damage response and associated pathways via non-covalent interactions

UBL5 associates non-covalently with the spliceosome to regulate splicing of proteins important for genome stability, including Sororin, XRCC3 and FANCI. (1) UBL5 promotes genome stability by ensuring proper expression of Sororin, a key component of the cohesin complex important for maintaining sister chromatid cohesion. (2) UBL5 promotes correct expression of XRCC3, a DNA damage response factor important for DNA double-strand break repair by homologous recombination (HR). UBL5 impacts on HR have not directly been assessed. In addition to maintaining FANCI expression via its splicing role (3), UBL5 interacts non-covalently and constitutively with FANCI to promote its stability, homodimerization, interaction with FANCD2 and monoubiquitylation, thereby promoting interstrand cross-link (ICL) repair by the Fanconi anemia (FA) pathway (4); Abbreviations: Ub, ubiquitin; XRCC3, X-ray repair cross-complementing factor 3.

Figure 5
UBL5 promotes DNA damage response and associated pathways via non-covalent interactions

UBL5 associates non-covalently with the spliceosome to regulate splicing of proteins important for genome stability, including Sororin, XRCC3 and FANCI. (1) UBL5 promotes genome stability by ensuring proper expression of Sororin, a key component of the cohesin complex important for maintaining sister chromatid cohesion. (2) UBL5 promotes correct expression of XRCC3, a DNA damage response factor important for DNA double-strand break repair by homologous recombination (HR). UBL5 impacts on HR have not directly been assessed. In addition to maintaining FANCI expression via its splicing role (3), UBL5 interacts non-covalently and constitutively with FANCI to promote its stability, homodimerization, interaction with FANCD2 and monoubiquitylation, thereby promoting interstrand cross-link (ICL) repair by the Fanconi anemia (FA) pathway (4); Abbreviations: Ub, ubiquitin; XRCC3, X-ray repair cross-complementing factor 3.

UBL5 directly regulates DNA interstrand cross-link (ICL) repair

In addition to pre-mRNA processing, UBL5 directly supports the functionality of DNA damage repair through the FA pathway, which is important for repairing ICLs [89]. To do so, UBL5 non-covalently binds to and stabilizes the central FA pathway component FANCI, thereby promoting key aspects of the FA pathway, including FANCI–FANCD2 heterodimerization as well as FANCD2 and FANCI monoubiquitylation (Figure 5) [89]. As a consequence, UBL5 promotes genome stability by contributing to the protection of cells from cytotoxic ICLs. In addition to FANCI, several DDR factors important for a range of DNA repair and associated pathways have been identified as putative UBL5 interactors (Table 1) [88], suggesting that additional DDR roles for UBL5 await discovery.

UFM1: the latest addition to the HR pathway

UFM1 (9.1 kD), the latest UBL to be identified [90,91], shares ∼17% of amino-acid sequence identity with ubiquitin and is conserved among vertebrates, but absent in yeast, which indicates roles specific to multicellular organisms (Figure 2B) [91,92]. The mature form of UFM1 contains a single conserved glycine residue used for substrate conjugation [91,92]. Thus, UFM1 differs from ubiquitin and other ligatable UBLs, which characteristically contain a di-glycine at their C-terminus (Figure 2A) [11]. UFMylation starts with the activation of mature UFM1 by an E1 called UBA5 [91,93]. The activated UFM1 is then transferred to a single known E2 enzyme, UFC1 [91,94], and finally, with the help of UFL1, the only E3 ligase known to date, UFM1 is conjugated to substrates [95]. Interestingly, UFL1 lacks an active cysteine and may thus function as a scaffold E3, reminiscent of RING-type ubiquitin-E3 ligases [95]. In addition, an E4 may contribute to UFMylation (UfBP1, also known as DDRGK1) (Table 1) [96–98]. UFM1 can also UFMylate itself on K69 [95], generating polyUFM1 topologies analogous to ubiquitin chains and other known polyUBL topologies [9,11]. Moreover, UFMylation of substrates can be reversed by the action of a recently identified subfamily of cysteine proteases that comprise UfSP1 and UfSP2 (Table 1). Between the two, UfSP2 is the main deUFMylase in humans [99], and also carries out the UFM1 precursor-processing step (Figure 2B). The UFM1 cascade has been linked to a wide variety of functions including homeostasis of the endoplasmic reticulum (ER), haematopoiesis, fatty acid metabolism, G-protein coupled receptor biogenesis, transcriptional control, autophagy, neurodevelopment, intestinal homeostasis and liver development [92,100]. Furthermore, several DDR factors have been identified as potential UFM1 substrates or interactors (Table 1) [101,102].

UFM1 plays important roles for DSB repair by HR

Recently, direct roles for UFMylation at sites of DNA damage have been uncovered (Figure 6). UFL1 appears to be recruited to DSBs, where it interacts with, and UFMylates K281 [103] and K282 of MRE11 [104]. MRE11 is a member of the MRN complex, which is required for activating ATM, one of the apical kinases crucial for initiating and amplifying the DDR (Figure 1). UFMylation of MRE11 is reported to be important for the recruitment of the MRN complex to DSBs, therefore facilitating DSB repair by HR. As a consequence, UFMylation phenocopies key functions of MRN such as optimal ATM activation, DSB repair by HR and overall maintenance of genome stability. In line with these findings, an MRE11 mutation (G285C) identified in uterine endometrioid carcinoma exhibits the same cellular phenotype as the UFMylation-defective MRE11 mutant (K282R) [103]. In addition to MRE11, histone H4 is UFMylated at K31 in response to DNA damage, which promotes the recruitment of the histone methyltransferase SUV39H1 and the acetyltransferase TIP60 (also known as KAT5) to DSBs, both of which are important for ATM activation. In a positive feedback loop, ATM phosphorylates UFL1 at S462, which enhances its E3 ligase activity and further reinforces ATM activation (Figure 6) [102].

UFMylation promotes ATM activation to facilitate DSB repair by homologous recombination

Figure 6
UFMylation promotes ATM activation to facilitate DSB repair by homologous recombination

UFMylation of MRE11 is important for formation of the MRE11-RAD50-NBS1 (MRN) complex under unperturbed conditions and ATM activation (1). Upon DNA damage, the MRN complex interacts with the UFM1-E3 ligase, UFL1, which mediates UFMylation of histone H4 (2). H4 UFMylation promotes DNA double-strand break (DSB) recruitment of the methyltransferase SUV39H1 (3), which trimethylates lysine 9 of histone H3 (H3K9me3) (4), followed by binding of the acetyltransferase TIP60 (also known as KAT5) to H3K9me3 (5). Combined with phosphorylation-mediated activation by c-Abl (6), TIP60 acetylates ATM (7), and promotes ATM activation together with its autophosphorylation (8) and interaction with MRN. Activated ATM phosphorylates histone H2AX at serine S139, which amplifies the DNA damage response to promote DSB repair by homologous recombination (9). Activated ATM also phosphorylates UFL1 at serine S462 (10), which enhances UFL1 activity and further amplifies ATM activation in a positive feedback loop (11). The exact sequence of some of the events is unknown; Ac: acetylation; M: methylation; P: phosphorylation.

Figure 6
UFMylation promotes ATM activation to facilitate DSB repair by homologous recombination

UFMylation of MRE11 is important for formation of the MRE11-RAD50-NBS1 (MRN) complex under unperturbed conditions and ATM activation (1). Upon DNA damage, the MRN complex interacts with the UFM1-E3 ligase, UFL1, which mediates UFMylation of histone H4 (2). H4 UFMylation promotes DNA double-strand break (DSB) recruitment of the methyltransferase SUV39H1 (3), which trimethylates lysine 9 of histone H3 (H3K9me3) (4), followed by binding of the acetyltransferase TIP60 (also known as KAT5) to H3K9me3 (5). Combined with phosphorylation-mediated activation by c-Abl (6), TIP60 acetylates ATM (7), and promotes ATM activation together with its autophosphorylation (8) and interaction with MRN. Activated ATM phosphorylates histone H2AX at serine S139, which amplifies the DNA damage response to promote DSB repair by homologous recombination (9). Activated ATM also phosphorylates UFL1 at serine S462 (10), which enhances UFL1 activity and further amplifies ATM activation in a positive feedback loop (11). The exact sequence of some of the events is unknown; Ac: acetylation; M: methylation; P: phosphorylation.

UFM1 and telomere maintenance

In addition to the above, UFMylation has been implicated in preventing telomere shortening, as highlighted by increased telomere attrition in HeLa cells knocked out for UFL1 and haematopoietic zebrafish cells deleted for ufm1 or ufl1. Interestingly, mutation of the UFMylation sites identified for MRE11 abrogated MRE11 interaction with the telomere protein TRF2, which could explain the significant reduction in telomere length observed. In contrast with the above-mentioned studies [102,103], no defects in DSB repair regarding MRN complex formation, ATM activation and HR were detected, potentially due to differences in the cell lines used, pointing towards variable requirements for UFMylation for different DDR pathways [104].

Conclusions and perspectives

Recent years have illustrated that UBLs play much wider-ranging roles in the DDR than anticipated. Moreover, proteomic studies point towards numerous additional DDR targets for covalent or non-covalent interactions with the above UBLs [36–39,69–71,88,101,102]. This wealth of data suggests that we have only started to uncover UBL functions in the DDR and associated pathways (Table 1). Future studies are required to validate these targets and help shed light on how exactly UBLs regulate different DNA repair and signalling pathways in normal contexts as well as in disease settings. Towards this aim, using and developing novel chemical tools, such as affinity-based probes and proximity labelling-based proteomics may prove useful [105]. Interestingly, chromatin remodelling components appear to be targeted by all of the UBLs discussed (Table 1). Given the large number of potential substrates contributing to chromatin remodelling, further validation is required to uncover the true extent to which UBLs impact on chromatin structure/function, and whether such effects directly regulate the DDR or have more indirect epigenetic functions.

To shed light on the mechanisms underlying UBL functions in the DDR, it will be key to identify the downstream UBL receptors that decode UBLylations and translate them into biochemical functions, an area that remains understudied both in the ubiquitin and UBL fields. In this regard, it is notable that the surface charge distribution can considerably differ between UBLs and ubiquitin (Figure 2A) [50,91], indicating the presence of distinct sets of interactors/downstream receptors and thus, the ability for separation-of-function at that level. For some UBLs, formation of chains has been demonstrated [28,97]: for instance, ISG15 can form mixed chains with K29 on ubiquitin [28], and polyUFM1 chains can be generated via linkage of K69 [97]. However, how exactly these topologies compare to single monoUBLylations, multiple monoUBLylations on individual proteins, and other mixed UBL chains will be important questions to answer in the future. If and how such mixed ubiquitin/UBL architectures may fine-tune the DDR, as demonstrated for instance for mixed ubiquitin/SUMO chains recognized by RAP80, a DDR factor important for DSB repair [106], remain other exciting aspects to uncover.

Compared with ubiquitin, UBLylations rely on a small number of enzymatic components identified to date (Figure 2B), posing the question on how these systems achieve specificity. Thus, one might speculate that additional UBLylation components, such as E3s, still await discovery. Differential tissue expression of UBLylation component/substrates and regulation of their expression, subcellular localization, level of activity etc. by distinct cellular stressors might form other regulatory layers that conform specificity. For instance, the low expression levels of ISGylation components under normal physiological conditions are considerably increased upon stimulation by inflammatory cytokines or DNA-damaging agents [32,35,40–42]. Such stimuli generate distinct banding patterns of ISG15 conjugates in immunoblots, implying that ISG15 can interact with specific sets of proteins in response to different external triggers [35]. Interestingly, immune signalling can be triggered by several components of the DDR including DNA damage sensors, transducer kinases, and effectors. Moreover, defects in the DDR are associated with susceptibility to infections or inflammatory diseases [107]. Indeed, much progress has recently been made in linking the immune response to the DDR. Given the established immune regulatory roles of ISG15, it will be exciting to discover in depth if and how ISGylation pathways that modulate the DDR may intertwine with immune responses.

Various UBLs as well as other PTMs act on p53 and PCNA, suggesting that these factors represent hubs for PTM regulation of the DDR. Understanding this cross-talk better e.g. to what extent such UBLylations overlap with each other and other PTMs is crucial for further enhancing our knowledge of the DDR. It will also be interesting to investigate if these modifications are in competition with each other, form compensatory mechanisms, occur in parallel or sequentially, and whether similar mechanisms hold true for other commonly targeted substrates. Given the broad effects of UBLs on cellular activities, it is not surprising that their deregulation is associated with a wide array of human diseases including cancer [43,45,60,63,65,72,79,97]. Thus, altered UBL levels can impact on the response of cancers to chemotherapeutics and vice versa. For example, several chemotherapeutic drugs increase ISGylation levels, and high levels of ISG15 conjugates have been reported in many primary tumours [43,45]. In addition, FAT10 plays a role in chemoresistance and is strongly overexpressed in several cancers with potentially oncogenic functions [60,63,65,72,79]. UFMylation has also been linked to cancer and may either promote or suppress tumorigenesis depending on the context [97,100]. While the focus of the present review is on DDR-related phenotypes, we refer the reader to the following reviews for additional information on in vitro and in vivo phenotypes associated with interventions of the UBLs discussed [31,72,100,108].

UBLylations are brought about by a series of conjugating enzymes (Figure 2B). Moreover, most of the UBLylations discussed in the present review, apart from FAT10, have been demonstrated to be reversible, and rely on the action of a single or a small set of specific deconjugating enzyme(s) (Figure 2B). As enzymes, these regulatory components are attractive for small molecule drug targeting, and the recent success of DNA repair inhibitors as anti-cancer therapies [109,110] highlights their potential as novel anti-cancer drug targets. In this regard, it is noteworthy that first-generation specific inhibitors of ubiquitin deconjugating enzymes have been developed and are currently approaching clinical trials [110,111], emphasizing the promise of extending equivalent approaches to UBLs. In conclusion, further research into validating and extending the roles of UBL cascades in the DDR is essential for enhancing our understanding of the molecular impacts of UBLylations on various human diseases and may eventually lead to new therapeutic avenues against them.

Summary

  • UBLs such as ISG15, FAT10, UBL5 and UFM1 are emerging as important co-regulators of a wide array of DDR functions.

  • Identification and characterization of additional UBLylation components and interactors/targets relevant for the DDR is required to shed further light on the extent to which UBLs modulate different aspects of the DDR.

  • Defining the downstream receptors that decode UBLylations into biochemical activities is crucial for unravelling the underlying mechanisms-of-action by which UBLs regulate different DDR pathways.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

We thank members of the Genome Stability Lab, particularly Zac Sandy, for useful discussions and acknowledge funding from a University of Manchester President’s Doctoral Scholar Award and BBSRC David Phillips Fellowship [grant number BB/N019997/1].

Abbreviations

     
  • ATG

    autophagy-related protein

  •  
  • BER

    base excision repair

  •  
  • DDR

    DNA damage response

  •  
  • DSB

    DNA double-strand break

  •  
  • FA

    Fanconi anemia

  •  
  • FAT10

    F locus adjacent transcription 10

  •  
  • FBR-MuSV

    Finkel-Biskis-Reilly murine sarcoma virus

  •  
  • HR

    homologous recombination

  •  
  • ICL

    interstrand cross-link

  •  
  • IR

    ionizing radiation

  •  
  • ISG15

    interferon-stimulated gene 15

  •  
  • MMR

    mismatch repair

  •  
  • NER

    nucleotide excision repair

  •  
  • NHEJ

    non-homologous end-joining

  •  
  • PIKK

    phosphoinositide 3-kinase-like kinase

  •  
  • PTM

    post-translational modification

  •  
  • SSA

    single-strand annealing

  •  
  • TLS

    translesion synthesis

  •  
  • UBL

    ubiquitin-like protein

  •  
  • Ub

    ubiquitin

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