Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway

Abstract

The maintenance of genome stability is critical for survival, and its failure is often associated with tumorigenesis. The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links (ICLs), and a germline defect in the pathway results in FA, a cancer predisposition syndrome driven by genome instability. Central to this pathway is the monoubiquitination of FANCD2, which coordinates multiple DNA repair activities required for the resolution of ICLs. Recent studies have demonstrated how the FA pathway coordinates three critical DNA repair processes, including nucleolytic incision, translesion DNA synthesis (TLS), and homologous recombination (HR). Here, we review recent advances in our understanding of the downstream ICL repair steps initiated by ubiquitin-mediated FA pathway activation.

Figure 1.

Interaction of the 15 FA proteins in a common ICL repair pathway in S phase. (A) Two replication forks converge on the DNA ICL that covalently links the two strands of DNA. (B) The FANCM–FAAP24–MHF1/2 complex recognizes the stalled replication fork structure and recruits the FA core complex to the ICL region. The translocase activity of FANCM prevents the collapse of replication fork independent of FA pathway activation. FANCM also initiates ATR-CHK1-dependent checkpoint response, which in turn phosphorylates multiple FA proteins, including FANCA/E/D2/I. (C) The FA core complex, a ubiquitin E3 ligase, monoubiquitinates FANCD2 and FANCI, and the ID heterodimeric complex is recruited to the DNA lesion. (D) FANCD2-Ub acts as a platform to recruit multiple nucleases to coordinate nucleolytic incisions flanking the ICL. FANCP/SLX4, which interacts with ERCC1–XPF and MUS81–EME1 structure-specific nucleases, and FAN1 5′-flap endonuclease are good candidates for this process. Both SLX4 and FAN1 contain the UBZ4 UBM essential for FANCD2-Ub-dependent recruitment to the DNA lesion. (E) Unhooking leaves cross-linked nucleotides tethered to the complementary strand, which is bypassed by TLS, mediated by specialized TLS polymerases such as REV1 and Pol ζ. (F) Incision creates a DSB, which is repaired by HR. Downstream FA proteins promote RAD51-dependent strand invasion and the resolution of recombinant intermediates. NER removes remaining adducts and fills the gap. (G) The USP1–UAF1 DUB complex removes monoubiquitin from FANCD2-I and completes the repair.

Figure 2.

UBZ is a signature domain of DNA repair proteins. Various amino acid sequences of known UBZ domains are shown, and their relative positions are marked as a red bar in the protein schematic. Amino acid residues are colored according to their physicochemical properties, and the conserved residues that comprise a zinc-binding core are shaded in the red box. UBZ domains are defined as a C2H2 (UBZ3) or C2HC (UBZ4) zinc finger module, and these two classes appear in DNA damage response proteins related to ICL repair (the FA pathway) and post-replication repair (PRR) (TLS). The major difference between the two domains is the fourth zinc ligand residue, which is a cysteine in the UBZ4 motif instead of histidine in the UBZ3. In addition, the aspartate residue within the H2 zinc-binding dyad of the UBZ3 (indicated as asterisk) is not absolutely conserved in the UBZ4 group, although alanine substitution was shown to disrupt the interaction with ubiquitin in WRNIP (Crosetto et al. 2008). In FAAP20, the aspartate residue outside the HC zinc-binding dyad (underlined) is also required for binding to ubiquitin, possibly compensating for the lack of aspartate in the conserved position (Ali et al. 2012). PCNA-Ub and FANCD2-Ub were proposed as primary targets for some of these UBZ domains, but several in vitro data also suggest that these UBZ domains are able to bind to the Lys 48- or Lys 63-linked polyubiquitin chains. The main function of the UBZ domain is to recruit UBZ-containing DNA repair factors to the site of DNA lesion by recognizing DNA damage-specific ubiquitin conjugation, including monoubiquitinated targets or polyubiquitin chains.

Figure 3.

Regulation of TLS in replication-associated ICL repair. The FA core complex not only regulates the incision step by monoubiquitinating FANCD2, but also contributes to the recruitment of a TLS polymerase REV1 to the lesion, thus promoting bypass of the ICL intermediate. FAAP20 is an important factor in both steps, as it stabilizes FANCA (therefore keeping the integrity of the FA core complex) and interacts with monoubiquitinated REV1 via its UBZ4 domain to promote stable association of the PCNA/REV1 DNA damage bypass complex at the stalled replication fork. REV1 controls the polymerase switching of TLS polymerases to restore the leading strand synthesis mediated by Pol ζ. It is not clear whether RAD18-dependent PCNA monoubiquitination is a prerequisite for the REV1 recruitment to the ICL region. In contrast, PCNA-Ub enhances the recruitment of UBZ4-containing SNM1A to the ICL site. The 5′–3′ exonuclease activity of SNM1A trims the unhooked intermediate to generate a favorable substrate for TLS (Wang et al. 2011).

Figure 4.

The FANCI–FANCD2 (ID) complex is targeted by USP1–UAF1. (A) The structure of the mouse ID complex generated by PyMOL software using Protein Data Bank ID 3S4W is shown where a trough-like shape of FANCI (pink) and FANCD2 (blue) are juxtaposed in an anti-parallel manner (schematic on top). The monoubiquitination sites of FANCD2 (Lys 559) and FANCI (Lys 522) are embedded in the ID interface just wide enough to create a tunnel to accommodate the C-terminal ubiquitin tail (shown in red). FANCI contains S/TQ clusters following the monoubiquitination site, and FANCI phosphorylation promotes FANCD2 monoubiquitination (shown in gray). FANCD2 and FANCI may be monoubiquitinated as a monomer, which increases the affinity for chromatin and allows heterodimerization at the DNA lesion. Ubiquitination may stabilize the heterodimerization and induces a conformational change that can fully expose the modified lysine for recruiting DNA repair factors. Alternatively, the ID complex may be loosely associated with chromatin, and ubiquitination may open the ID interface, relocating the ID complex to damage-induced foci at the lesion. FANCD2 and FANCI by themselves have been shown to recognize several DNA structures. Thus, the ubiquitinated ID complex at the ICL may adopt a different conformation compared with the one modeled here. Of note, the SIM of FANCI is exposed outside (shown in green), allowing the targeting of USP1–UAF1 via the SLD2 domain of UAF1. The interaction may trigger the exposure of the ubiquitinated lysine to provide access to the USP1 active site for isopeptide cleavage. (B) By heterodimerizing with FANCD2, FANCI regulates both FANCD2 ubiquitination and deubiquitination through phosphorylation and SIM-mediated DUB targeting, respectively.