**5. DUBs and the DDR**

Cells undergo DDR to sense and repair unique lesion structures in the damaged DNA. E fficient DDR protects cells from genomic instability [121,122]. Ubiquitination regulates DDR by controlling DDR protein localization, activity, and stability [123]. DUBs play critical roles in di fferent stages of the DDR through the regulation of many molecules involved in DNA repair (Figure 3). DNA repair is important for preventing tumor formation [124]. Proliferating cell nuclear antigen (PCNA) is a key molecule that mediates the tolerance to DNA damage and allows the growth of tumors. PCNA is monoubiquitinated in response to DNA damage. A fission yeas<sup>t</sup> study showed the importance of UBP2, UBP12, and UBP15 in the stabilization of mono, di, and polyubiquitylated forms of PCNA, which sensitize cells to DNA damage [69]. In addition, PCNA can be deubiquitinated by USP1 in the crosslink repair pathway in Fanconi anemia [71–73]. In a complex with its cofactor UAF1, USP1 reverses PCNA ubiquitination [74]. UCHL5 regulates double-strand break (DSB) resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation [70]. In addition, USP20 plays role in genome maintenance and DNA repair by enhancing recombinational repair of collapsed replication forks [125]. Furthermore, USP9X regulates the DNA checkpoint protein claspin during S phase, suggesting a role in DNA repair [79]. USP7-promoted PHF8 stabilization confers cellular resistance to genotoxic insults and is required for the recruitment of BLM and KU70, which are both essential for DNA DSB repair [36].

**Figure 3.** Roles of DUBs in DNA damage response. Various DUBs (light purple) have been shown to interact with molecules (various colors) that play roles in DNA repair and chromosomal stability during DNA damage. Proliferating cell nuclear antigen (PCNA) plays important roles during DNA replication and repair, while BRCA members are the key players in repairing the DNA lesions such as DNA double-strand breaks. In addition, BLM repairs DNA double-strand breaks to maintain genome stability. Detailed information can be found in the main text.

Breast-cancer susceptibility gene (BRCA) 1 contributes to DNA repair and the maintenance of chromosomal stability in response to DNA damage [126]. BRCA1 appears to play roles in two distinct pathways of DSB repair, non-homologous end joining and homology-directed repair, through the regulation of different effectors. It has been reported that several DUBs can regulate BRCA1. The BRCA1-associated DUB BAP1 is mutated in mesothelioma and melanoma [65]. BAP1 is a phosphorylation target for the DDR kinase ATM, and BAP1 mediates rapid poly(ADP-ribose)-dependent recruitment of the polycomb DUB complex PR-DUB to repair DNA DSBs [65]. In addition, both cezanne (OTUD7B) and cezanne2 (OTUD7A) promote the recruitment of the Rap80/BRCA1-A complex by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin in response to DNA repair [68]. Another DUB, USP11, forms a complex with BRCA2. It deubiquitylates the partner and localizer of BRCA2 to enhance DNA repair [75]. BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lys63-specific DUB involved in the DDR. BRCC3 inactivation increases the release of several cytokines, including G-CSF, which enhances proliferation in AML cell lines [127]. Further, OTUD5, a specific stabilizer of the UBR5 E3 ligase, is reported to localize at DNA DSBs. OTUD5 plays two roles at DSBs. First, OTUD5 interacts with UBR5 and represses RNA Pol II-mediated elongation and RNA synthesis. In addition, OTUD5 interacts with the FACT component SPT16 and antagonizes histone H2A deposition at DSBs [67].

Histone ubiquitination at DNA breaks is required for activation of the DDR and DNA repair. BRCA1-BARD1-catalyzed ubiquitination of histone H2A primes chromatin for repair by homologous recombination during the DDR. Ubiquitination of histone H2A and γH2AX by the UBQ ligases RNF168 and RNF8 generates a cascade of ubiquitination. USP3 deubiquitinates ubiquitinated γH2AX and H2A [76]. USP48 is another H2A DUB that is specific for the C-terminal BRCA1 ubiquitination site. USP48 promotes genomic stability by antagonizing the BRCA1 E3 ligase function. Depletion of USP48 increases the distance between p53-binding protein 1 (53BP1) from the DNA break point [77]. It should be noted that histone ubiquitination by RNF168 is a critical event for the recruitment of BRCA1 and 53BP1, and the stability of RNF168 can be regulated by USP7. Depletion of USP7 impairs H2A and γH2AX monoubiquitination, leading to decreases in the levels of pBmi1, Bmi1, RNF168, and BRCA1 under ultraviolet radiation-induced DNA damage [78]. Moreover, USP3, a histone H2A DUB, negatively regulates UBQ-dependent DDR signaling through regulation of chromatin ubiquitination in response to genotoxic stress [128]. Lastly, CYLD deubiquitinates p53 and facilitates its stabilization in response to genotoxic stress. Loss of CYLD catalytic activity causes impaired DNA damage-induced p53 stabilization and activation of skin tumorigenesis [66].
