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Review
. 2024 Apr 10;481(7):515-545.
doi: 10.1042/BCJ20230284.

DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability

Benjamin M Foster  1 , Zijuan Wang  1 , Christine K Schmidt  1
Affiliations
Review

DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability

Benjamin M Foster et al. Biochem J. .

Abstract

Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.

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Conflict of interest statement

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

Figures

Figure 1.
Figure 1.. Circular plot of the human deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) currently known.
Enzymes are grouped by major DUB families and proteases against UBLs. DUBs with additional ULP activity (e.g. USP18 and USP36) are annotated with the appropriate UBL outside of the circle. DUBs and ULPs referenced in this review to have a role in the DNA damage response (DDR) (Table 1) are highlighted in bold with a filled background. CSN5 and COPS5 (indicated by an *) are the same protein duplicated due to its annotation as a JAMM-family protease while having deNEDDylation activity. DEN1 is also known as NEDP1 or SENP8. ZUP1 is so far the only member of this DUB family and only a single member of USP17 (USP17L1) is shown due to its highly polymorphic copy number variation. ATG4A-D are cysteine proteases related to autophagy signalling pathways.
Figure 2.
Figure 2.. Overview of DUBs involved in DSB repair signalling pathways.
(A) Upon DNA damage causing a DSB (red star), a plethora of signalling events involving Ub can occur to regulate the repair pathway choice in the context of other cell signals, such as cell cycle cues. MDC1 (SENP2 and USP7), TIP60 (USP7), RNF168 (USP34 and USP7), and RAP80 (USP13) are all stabilised through the action of DUBs to counteract polyUb-mediated protein degradation. RAP80 can bind both Ub and SUMO signals upon DSB formation to recruit BRCA1-A complex components, enabling sequestration of BRCA1-BARD1 and localisation of K63-linked polyUb DUB activity of BRCC36. Other DUBs, such as OTUB2, POH1, USP26, USP37, and USP44, dampen RNF168-mediated polyUb chain formation ensuring proficient repair of the DSB. USP51 can directly bind H2AK13/15-Ub-containing nucleosomes to remove the Ub signal, with USP3 and USP16 also implicated to remove H2A(X)-Ub modifications. USP11 and BAP1 can remove the polycomb-related H2AK119-Ub mark, likely to regulate transcription during DDR signalling and local chromatin accessibility, further aided by INO80 stabilisation through the DUBs BAP1 and UCHL5. (B) H2AK13/15-Ub combined with H4K20me2 is bound by 53BP1 in a multidentate manner to recruit the shieldin complex to help protect DNA ends and displace the HR repair machinery. Ku80 chromatin retention is regulated by the DUBs OTUD5 and UCHL3. (C) During S/G2 phase, the Ub E3 ligase BRCA1-BARD1 can bind H2AK13/15-Ub combined with unmethylated H4K20 to ubiquitylate H2AK125/127/129 to promote HR-mediated repair signalling. The DUB USP48 can counteract this Ub signal to prevent hyper-resection. The MRN complex and CtIP are stabilised at sites of DSBs through the action of USP4. The BRCA1-PALB2-BRCA2 complex, required for BRCA2-mediated loading of RAD51, is stabilised by cell cycle-regulated USP11, which removes an inhibitory Ub that otherwise disrupts the BRCA1-PALB2 interaction during S/G2 phase. DUB/ULP activity is represented by dashed arrows.
Figure 3.
Figure 3.. Overview of DUBs in DDR signalling pathways associated with replication stress, NER, and BER.
(A) DUBs act on central signalling nodes within the DDR signalling pathways upon replication stress. PCNA is mono-ubiquitylated by RAD6/RAD18 upon stalling or slowing of the replication fork due to DNA damage. Mono-ubiquitylated PCNA forms a platform to recruit more error-prone TLS polymerases to bypass the lesion. RAD18 and the TLS polymerases are themselves protected from polyUb-mediated degradation via the proteasome due to the action of USP7. To restore normal replicative processes, the mono-Ub is removed by USP1-UAF1. Template switching (TS) is thought to be instigated by HLTF- and SHPRH-dependent poly-ubiquitylation of PCNA, recruiting the helicase and endonuclease ZRANB3 as the downstream effector. Replication stalling can lead to excessive ssDNA bound by ATR phosphorylated RPA, which can lead to replication fork reversal or TLS via RFWD3-dependent poly-ubiquitylation of surrounding proteins, including of PCNA and RPA. ZUP1 is hypothesised to be a candidate DUB to counteract polyUb chain formation during this process. Interstrand cross-links (ICLs) are generally repaired via the Fanconi anaemia (FA) repair pathway. FANCI-D2 is ubiquitylated by the FA core complex in an ATR-dependent manner, resulting in the FANCI-D2 heterodimer forming a DNA clamp at the site of damage to facilitate downstream repair. USP1-UAF1 can remove the mono-Ub mark. SENP6 is a deSUMOylase that counteracts polySUMO2/3 chain formation on FANCI-D2, preventing RNF4-mediated poly-ubiquitylation leading to VCP/p97-mediated extraction of FANCI-D2 from chromatin. One mechanism to remove DNA-protein cross-links (DPCs) is through the DNA-dependent metalloprotease SPRTN whose activity requires removal of an inhibitory mono-Ub by USP7 and/or VCPIP1. BAP1 stabilises the chromatin remodeller INO80 through its DUB activity to provide a DNA repair-proficient chromatin environment upon damage. (B) DUBs in nucleotide excision repair (NER) primarily protect repair factors from degradation. CUL4-DDB2 forms an E3 ligase for the process, with USP24 and USP44 thought to stabilise DDB2 by removing polyUb chains. One of the substrates for CUL4-DDB2 is XPC, which is protected from chromatin extraction and degradation by the action of USP7, with USP7 also removing polyUb from CSA via UVSSA-mediated recruitment. The endonuclease, XPF-ERCC1, is retained at sites of damage by USP45. (C) During base excision repair (BER), Pol β abundance and chromatin engagement is regulated through Ub signalling by the E3 ligases Mule and CHIP, with the DUB USP47 able to remove the polyUb signal. Repair of DNA base alkylation by ALKB2 and ALKB3 is enabled by a DUB complex made up of USP9X and USP7, with OTUD4 forming a non-catalytic scaffold subunit. DUB/ULP activity is represented by dashed arrows.
Figure 4.
Figure 4.. Overview of the role of ULPs in DDR signalling pathways.
(A) The role of SENPs (SUMO ULPs) and the CSN (NEDD8 ULP) in DSB repair signalling. SENP6 is required to regulate SUMO2/3 levels on critical factors for DSB repair such as BRCA1-BARD1, 53BP1, BLM, XPF-ERCC1, and RPA to maintain active levels in an RNF4- and VCP/p97-dependent manner. SENP7 can deSUMOylate KAP1 to release its interaction with the chromatin remodeller, CHD3, which, along with phosphorylation by ATM, ensures chromatin relaxation in response to DSBs within heterochromatin regions. SENP2 and ATXN3 regulate the chromatin retention of MDC1 upon DNA damage in an RNF4- and VCP/p97-dependent manner, ensuring DDR signalling to repair the damage. The COP9 signalosome (CSN) is recruited to DSBs in a NEDD8-dependent manner, with CSN3 phosphorylated by ATM. CSN can remove NEDD8 modifications from Cullin-RING ligases (CRLs), histones, and other proteins during the DDR. (B) UFMylation of MRE11 and histone H4 in response to DNA damage occurs via the E3 ligase UFL1, which is recruited to DSBs through an interaction with the MRN complex at DSBs. H4 UFMylation enables downstream recruitment of the acetyltransferase TIP60 (via SUV39H1-mediated H3K9me3 formation), which acetylates ATM thereby promoting its activation and interaction with MRN. ATM kinase phosphorylation of itself and H2AX at S139 amplifies the DDR signalling for repair by HR. ATM-mediated phosphorylation of S462 on UFL1 further enhances UFL1 activity, resulting in a positive feedback loop. Recruitment of one of the two known UFM1 ULPs, UfSP2, to DSBs is prevented by inhibitory ATM-mediated phosphorylations at its S374/S381 residues, which are removed by the WIP1 phosphatase, allowing UfSP2 to bind MRN to deUFMylate H4 and dampen the ATM DDR signal upon successful repair. (C) During S phase, DNA damage blocking or stalling of the replication machinery leads to mono-ubiquitylation of PCNA at K164 by RAD6/RAD18, resulting in recruitment of more error-prone Y-family TLS polymerases and bypass of the lesion. To recycle unmodified PCNA and resume normal replicative processes, the E3 ligase EFP/TRIM25 can attach ISG15 to K168 on PCNA, leading to USP10-mediated removal of the mono-Ub on PCNA. EFP/TRIM25 can ISGylate K164, likely to prevent re-addition of Ub, before both ISG15 modifications are removed by USP18. How this mechanistically works in parallel with the established action of USP1-UAF1 is unknown. DUB/ULP activity is represented by dashed arrows.
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