*4.2. Deficiency of FUS or Mutations of FUS A*ff*ect the Repair of DNA Strand Breaks*

Because FUS deficiency increases genome instability in animal and cell models, FUS has been the subject of further research on its role in the repair processes. As mentioned above, eukaryotic cells have evolved two pathways to repair DSBs, namely, HR and NHEJ (Figure 3a,b) [86,90]. Other experimental pieces of direct evidence for the participation of FUS in DSB repair come from examination of the effects of a knockdown of FUS by a small interfering RNA or expression of fALS-associated mutant FUS versions (R244C, R514S, H517Q or R521C) in murine primary neuronal culture and/or human osteosarcoma U2OS cells [37]. Upon DNA damage, FUS depletion leads to a decrease in the level of H2AX phosphorylation, an impairment of DSB repair foci creation and deficient accumulation of DDR proteins such as p53-binding protein 1 (53BP1), Nijmegen breakage syndrome 1 (NBS1), phospho-ATM (ataxia telangiectasia mutated) and Ku70 (Figure 3a,b). The role of FUS in DSB repair is further underscored by results of FUS depletion: decreased HR and NHEJ efficiency and elevating the number of DNA damages in primary neuronal culture [37]. Besides, FUS directly associates with chromatin [91] and with a remodelling factor such as histone deacetylase 1 (HDAC1) [37]. HDAC1 plays an important part in the promotion of DNA DSB repair in post-mitotic neurons [92]. Furthermore, the expression of FUS mutants defective in their interaction with HDAC1 impairs both NHEJ and HR pathways in the U2OS−GFP cell line [37]. These observations indicate an impaired capacity to repair a DNA DSB when expression levels of FUS are low or FUS is mutated in the cell. The functions of FUS in NHEJ and HR repair pathways are therefore clearly non-transcriptional.

In addition to its functions in DSB repair, FUS is involved in DNA SSB repair in HEK293 cells [40]. FUS co-immunoprecipitates with BER players such as XRCC1 and DNA ligase III (Lig III) and stimulates Lig III activity via direct interaction. Moreover, a CRISPR/Cas9-mediated knockout of FUS in HEK293 cells reduces the efficiency of SSB repair [40]. Defective DNA repair of oxidative DNA damage is observed in induced pluripotent stem cell lines derived from ALS patients carrying either the R521H or P525L mutation of FUS (Figure 1), which promotes the formation of cytosolic FUS aggregates at the expense of the pool of nuclear FUS [66,93].

These findings therefore define FUS as a novel participant of DNA break repair processes that plays an upstream role in DSB signalling; these data also point to a functional link to HR, NHEJ and BER/SSB repair via interaction with DNA intermediates and/or repair factors. In the case of DSB repair (Figure 3a,b), FUS facilitates the initial recruitment of DNA damage signalling proteins to DNA lesions and regulates HDAC's chromatin-remodelling activity [37]. In the case of SSB repair (Figure 4), FUS interacts with the XRCC1–DNA ligase III complex and stimulates the SSB ligation step [40]. On the other hand, we cannot rule out the possibility that FUS has functions downstream of ATM or DNA-PK in response to DNA breaks. This is because FUS phosphorylation drives its translocation to the cytoplasm under genotoxic stress thereby possibly affecting FUS functions in RNA metabolism [36,39].

#### *4.3. FUS Is Connected to DNA Strand Breaks by PARPs' Signalling Activities*

Although in vitro studies indicate that FUS directly binds to single- or double-stranded DNA or G-quadruplexes in telomeres [49,94], how FUS is recruited to the sites of DNA damage in the cell has been an open question. FUS recruitment to DNA repair sites has been analysed owing to the development of the laser micro-irradiation technique able to generate a spatially controlled DNA damage region in the cell nucleus [37,38,40]. The first evidence that FUS can be translocated directly to DNA damage foci was obtained in cells subjected to 405 nm laser micro-irradiation. Moreover, FUS recruitment to DSBs is specifically dependent on the activity of PARP1, thus pointing to an interaction between FUS and the PAR produced by PARP1 at DNA damage sites [51]. Later, PAR-dependent FUS accumulation at damaged-DNA sites has been detected in response to oxidative DNA damage induced by a UVA (320–400 nm) laser, suggesting the recruitment of FUS not only to DSBs but also to SSBs generated directly or indirectly during the repair of oxidised bases in DNA [38]. Therefore, DNA damage–dependent activation of PARPs and the synthesis of PAR are some of the ways in which FUS can be recruited to sites of DNA damage [37,38,40]. PARPs, primarily PARP1 and PARP2, can directly recognise damaged DNA, and their binding to it results in PARP activation [95,96]. PARP enzymes use NAD<sup>+</sup> as a substrate and catalyse the transfer of an ADP-ribose residue from NAD<sup>+</sup> to target protein-acceptors, leading finally to the synthesis of ADP-ribose chains (PAR) attached to proteins, mainly PARPs themselves [21] (Figure 5). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 8 of 18 development of the laser micro*-*irradiation technique able to generate a spatially controlled DNA damage region in the cell nucleus [37,38,40]. The first evidence that FUS can be translocated directly to DNA damage foci was obtained in cells subjected to 405 nm laser micro*-*irradiation*.* Moreover, FUS recruitment to DSBs is specifically dependent on the activity of PARP1, thus pointing to an interaction between FUS and the PAR produced by PARP1 at DNA damage sites [51]. Later, PARdependent FUS accumulation at damaged-DNA sites has been detected in response to oxidative DNA damage induced by a UVA (320–400 nm) laser, suggesting the recruitment of FUS not only to DSBs but also to SSBs generated directly or indirectly during the repair of oxidised bases in DNA [38]. Therefore, DNA damage–dependent activation of PARPs and the synthesis of PAR are some of the ways in which FUS can be recruited to sites of DNA damage [37,38,40]. PARPs, primarily PARP1 and PARP2, can directly recognise damaged DNA, and their binding to it results in PARP activation [95,96]. PARP enzymes use NAD+ as a substrate and catalyse the transfer of an ADP-ribose residue from NAD+ to target protein-acceptors, leading finally to the synthesis of ADP-ribose chains (PAR) attached to proteins, mainly PARPs themselves [21] (Figure 5).

PAR chains is commonly linked to Lys/Arg or Glu/Asp residues attached to the C1" or C1",C2", C3" atoms of ADP-ribose with the formation of ketoamine and carboxyl ester, respectively (Figure 5). Moreover, amino acid specificity of PARP1 (PARP2) from Glu/Asp to Ser residue can be changed under influence of other protein factors such as histone PARylation factor 1 [95,97]. PAR chains is commonly linked to Lys/Arg or Glu/Asp residues attached to the C1" or C1",C2", C3" atoms of ADP-ribose with the formation of ketoamine and carboxyl ester, respectively (Figure 5). Moreover, amino acid specificity of PARP1 (PARP2) from Glu/Asp to Ser residue can be changed under influence of other protein factors such as histone PARylation factor 1 [95,97].

**Figure 5.** A diagram of the structure of PAR and protein PARylation. The scheme illustrates chemical structure of NAD+ and a chain of ADP-ribose units linked by the α(2′-1′′) *O*-glycosidic bond between ribose residues (linear chain) and by the α(2′′-1′′′) glycosidic bond between two nicotinamideproximal ribose residues (branching). PARG and ARH3 is the main enzymes that degrade PAR and possesses exo- and endoglycosidase activities hydrolysing the glycosidic bonds between ribose units of PAR [98,99]. **Figure 5.** A diagram of the structure of PAR and protein PARylation. The scheme illustrates chemical structure of NAD<sup>+</sup> and a chain of ADP-ribose units linked by the α(2<sup>0</sup> -100) *O*-glycosidic bond between ribose residues (linear chain) and by the α(200-1000) glycosidic bond between two nicotinamide-proximal ribose residues (branching). PARG and ARH3 is the main enzymes that degrade PAR and possesses exoand endoglycosidase activities hydrolysing the glycosidic bonds between ribose units of PAR [98,99].

Protein PARylation is a reversible process mainly due to poly(ADP-ribose) glycohydrolase (PARG) activity and ADP-ribosyl-acceptor hydrolases 3 (ARH3), which catalyse the cleavage between ADP-ribose structural units at the terminal position and inside the polymer, thereby releasing ADP-ribose or oligo(ADP-ribose), respectively [98,99]. Thus, the action of PAR*-*degrading enzymes makes protein PARylation a dynamic and reversible post-translational modification and plays an important role in the regulation of DNA repair [98–100]. Protein PARylation, PAR length Protein PARylation is a reversible process mainly due to poly(ADP-ribose) glycohydrolase (PARG) activity and ADP-ribosyl-acceptor hydrolases 3 (ARH3), which catalyse the cleavage between ADP-ribose structural units at the terminal position and inside the polymer, thereby releasing ADP-ribose or oligo(ADP-ribose), respectively [98,99]. Thus, the action of PAR-degrading enzymes makes protein PARylation a dynamic and reversible post-translational modification and plays an important role in the regulation of DNA repair [98–100]. Protein PARylation, PAR length and formation

and formation of protein-free PAR molecules all contribute to the regulation of DNA repair, in

of protein-free PAR molecules all contribute to the regulation of DNA repair, in particular, through the binding of proteins to PAR or their PARylation [16,21,25,100]. Therefore, PAR (either attached to proteins or free), because of its biochemical properties and tight regulation of its synthesis and degradation by PARP and PARG/ARH3 activities, is considered a critical factor that orchestrates reversible assembly of DNA repair compartments [16,25,99,101]. It is commonly accepted now that PARPs and PARylation regulate DSB and BER/SSB repair [102,103]. Recent research showed that FUS binds to PAR non-covalently and/or can be PARylated in vivo and in vitro [17,26,27,40,101]. Consistently with this notion, the FUS interaction with PAR after DNA damage constitutes the missing link between FUS and DNA repair events [17,38,40,51,56,101]. In line with this view, PAR synthesis at DNA damage sites induces the relocation of FUS to DNA damage foci [17,38,40,51]. Furthermore, inhibition of PARP activity impairs FUS accumulation at sites of laser-induced damage in the cell, and it is likely that the absence of PAR prevents FUS from being directed to DNA damage sites [17,38,51].

Consequently, FUS may take part in DNA strand break repair in a PAR-dependent manner, although little is known about the functional significance of FUS–PAR interactions in DNA repair [40,104]. One supposition is the unusual capacity of FUS to form a liquid-like compartment making it an ideal organiser of DNA repair compartments [17,101,104] able to concentrate damaged DNA at the early stages of the DNA strand break response in order to undergo spatially controlled PAR-dependent phase separation [101,104].
