**5. The DNA Crosslinks Repair Deals with NER and FA Pathways with Implication for Cancer**

Inter-strand crosslinks (ICLs) covalently link the two strands of the DNA double helix and they arise from exposure to chemicals such as DNA damaging agents used in cancer therapy.

In quiescent cells (G0/G1), an ICL is recognized by NER pathway, which is also involved in the repair of intra-strand crosslinks and UV-damage [85] (Figure 4). In NER, two mechanisms of DNA damage sensing are known: one pathway recognizes damage all over the genome, and the other one recognizes damage in the transcribed strand of active genes that cause blockage of the RNA polymerase II. In the first case, detection is performed by the binding of the XPC complex (XPC, HR23B and CENT2) to the non-damaged strand, assisted by PARP1 activity [86]. In the second case, arrest of RNA polymerase II is the initial signal that promotes recruitment of the Cockayne syndrome group A (CSA) and group B (CSB) proteins. At this point, both pathways require XPA that acts as a scaffold organizing the other components of the pathway [87] and the transcriptional factor IIH complex (TFIIH) which first unwinds the DNA around the damage by the helicase activity of XPB and XPD. Then, DNA damage is removed by the excision repair protein ERCC-1-XPF and XPG endonuclease activity, generating a gap that is filled by DNA polymerase and by the DNA ligase activity of XRCC1-LIG3 or LIG1 [88].

In S phase, ICL lesions cause stalling of DNA replication forks that are recognized by the FA pathway (Figure 4). The anchor complex is the one recognizing the stalled replication fork; when activated, the anchor complex recruits a core complex which attaches a single ubiquitin to both FANCD2 and FANCI that together form the ID2 complex. This ubiquitination is essential for the activation of the nucleases involved in the unhooking of the DNA strands that is carried out by SLX4 and XPF [89], and also for the recruitment of the TLS polymerases that replace the stalled DNA polymerases and bypass the lesion via TLS; this pathway is promoted by ubiquitination of TLS regulator PCNA, which is performed by FANCD2 [90]. Among TLS polymerases, Pol ξ is the one involved in the lesion bypass step of ICL, while REV1 facilitates polymerase switching and coordinates extension steps [91]. FANCD2 deubiquitination is then performed by deubiquitinating complex USP1/UAF1. Finally, after DNA unhooking, the downstream players of the pathway coordinate HR to repair the DSB formed; these players are FANCS (BRCA1), FANCD1 (BRCA2), FANCN (PALB2), and FANCO (RAD51); they are referred to as downstream components of the FA pathway since their activation depends on the mono-ubiquitination step (Figure 4) [92].

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**Figure 4.** Bulky lesions are repaired by nucleotide excision repair, inter-strand crosslinks formed in S/G2 phase, are repaired by Fanconi Anemia pathway. (**A**) In NER, the bulky lesion is recognized by PARP1 and XPC; they recruit XPA that acts as a scaffold organizing the helicases XPB and XPD that unwind the DNA around the damage. Then, several bases are removed by ERCC-1-XPF and XPG endonuclease; the gap generated is filled by Pol-β and ligated by XRCC1, LIG3/LIG1. (**B**) Convergent arrested-replication forks are recognized by the anchor complex; this recruits the core complex which attaches a single ubiquitin residue to FANCI and FANCD2. After, SLX4 and XPF nucleases unhook the DNA strand determining a DSB on one strand. The strand without the DSB is repaired by TLS by Pol ξ assisted by REV1; the other strand is repaired via HR by FANCS (BRCA1), FANCD1 (BRCA2), FANCN (PALB2) and FANCO (RAD51). **Figure 4.** Bulky lesions are repaired by nucleotide excision repair, inter-strand crosslinks formed in S/G2 phase, are repaired by Fanconi Anemia pathway. (**A**) In NER, the bulky lesion is recognized by PARP1 and XPC; they recruit XPA that acts as a scaffold organizing the helicases XPB and XPD that unwind the DNA around the damage. Then, several bases are removed by ERCC-1-XPF and XPG endonuclease; the gap generated is filled by Pol-β and ligated by XRCC1, LIG3/LIG1. (**B**) Convergent arrested-replication forks are recognized by the anchor complex; this recruits the core complex which attaches a single ubiquitin residue to FANCI and FANCD2. After, SLX4 and XPF nucleases unhook the DNA strand determining a DSB on one strand. The strand without the DSB is repaired by TLS by Pol ξ assisted by REV1; the other strand is repaired via HR by FANCS (BRCA1), FANCD1 (BRCA2), FANCN (PALB2) and FANCO (RAD51).

Germline mutations of the FA-related genes determine development of FA which is a genetic disease causing an impaired response to DNA damage. The majority of the affected people develop cancer, most often leukemia [93]. FA disease can develop also by mutations of HR-related genes such as *BRCA1*, *BRCA2,* and *PALB2*; hence, the FA pathway is often called the FA/HR pathway. Alterations of FA-related proteins are also associated to breast and ovarian cancer development; Germline mutations of the FA-related genes determine development of FA which is a genetic disease causing an impaired response to DNA damage. The majority of the affected people develop cancer, most often leukemia [93]. FA disease can develop also by mutations of HR-related genes such as *BRCA1*, *BRCA2,* and *PALB2*; hence, the FA pathway is often called the FA/HR pathway. Alterations of FA-related proteins are also associated to breast and ovarian cancer development; mutations of

mutations of FANCM, a component of the anchor complex, and FANCA, which is part of the core

complex, are both correlated to increased risk of breast cancer [94].

FANCM, a component of the anchor complex, and FANCA, which is part of the core complex, are both correlated to increased risk of breast cancer [94].
