The ID2 complex, composed of FANCD2 and FANCI, binds to branched DNA structures, such as stalled replication forks at DNA interstrand crosslinks (ICL-DNA) (Yuan et al. 2009, Longerich et al. 2009, Joo et al. 2011). The ID2 complex also interacts with the FA core complex component FANCL, activating the E3 ubiquitin ligase activity of FANCL (Rajendra et al. 2014, Longerich et al. 2014). Up to fifty FANCD2 molecules (ID2 complexes) may be recruited per one ICL, probably spreading to surrounding DNA (Douwel et al. 2014). The E2 ubiquitin ligase UBE2T is recruited to ICL-DNA by binding to the FANCL subunit of the FA core complex independently of the ID2 complex (Machida et al. 2006, Alpi et al. 2007, Hodson et al. 2014).

FANCD2 and FANCI, the components of the ID2 complex, are monoubiquitinated at DNA interstrand crosslinks (ICL-DNA) by the coordinated action of the E2 ubiquitin ligase UBE2T and the E3 ubiquitin ligase FANCL (Machida et al. 2006, Alpi et al. 2007, Sims et al. 2007, Smogorzewska et al. 2007, Longerich et al. 2009, Sato et al. 2012, Hodson et al. 2014). FANCL achieves the maximal catalytic activity as part of the ICL-DNA-bound FA core complex, and requires the presence of at least FANCB and FAAP100 subunits of the FA core complex to monoubiquitinate the ID2 complex (Rajendra et al. 2014, Longerich et al. 2014). FANCD2 is monoubiquitinated on lysine residue K561, while FANCI is monoubiquitinated on lysine residue K523 (Alpi et al. 2008, Longerich et al. 2014). In the absence of FANCD2, a DNA-bound FANCI can be monoubiquitinated in a FANCL-independent manner (Longerich et al. 2014).

UBE2T is also monoubiqutinated by FANCL on lysine residues K91 and K182 during the process of ID2 monoubiquitination. Monoubiquitination of UBE2T may serve as a self-inactivating mechanism that negatively regulates the Fanconi anemia pathway (Machida et al. 2006).

FANCD2 monoubiquitination promotes stability of the ID2 complex and its retention at ICL-DNA, and enables recruitment of additional proteins that participate in the repair of ICL-DNA (Garcia-Higuera et al. 2001, Smogorzewska et al. 2007, Alpi et al. 2008, Joo et al. 2011).

In addition to FANCM, FAAP24, APITD1 (MHF1) and STRA13 (MHF2), the FA core complex also includes FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP20 and FAAP100 (Singh et al. 2010, Yan et al. 2010, Leung et al. 2012). While FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL, and probably FAAP20 and FAAP100, can assemble a complex in the nucleoplasm, they are unable to load onto DNA in the absence of FANCM and FAAP24 (Kim et al. 2008, Yan et al. 2010, Leung et al. 2012).

The complex of ATR and ATRIP (ATR:ATRIP) is recruited to replication forks blocked by DNA interstrand crosslinks (ICL-DNA) through interaction with the RPA complex and the Fanconi anemia (FA) core complex. The RPA heterotrimer associates both with single strand DNA (ssDNA) that is produced by DNA resection at ICL-DNA-stalled replication forks and with the FANCM and FAAP24 components of the FA core complex (Huang et al. 2010). ATRIP directly interacts with the FANCL component of the FA core complex (Tomida et al. 2013). The presence of RAD17 and TOPB1, which is required for ATR activation at DNA double strand breaks (DSBs), is not needed for ATR activation at ICL-DNA (Tomida et al. 2013).

Fanconi anemia (FA) is a genetic disease of genome instability characterized by congenital skeletal defects, aplastic anemia, susceptibility to leukemias, and cellular sensitivity to DNA damaging agents. Patients with FA have been categorized into at least 15 complementation groups (FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O and -P). These complementation groups correspond to the genes FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCJ/BRIP1, FANCL, FANCM, FANCN/PALB2, FANCO/RAD51C and FANCP/SLX4. Eight of these proteins, FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, together with FAAP24, FAAP100, FAAP20, APITD1 and STRA13, form a nuclear complex termed the FA core complex. The FA core complex is an E3 ubiquitin ligase that recognizes and is activated by DNA damage in the form of interstrand crosslinks (ICLs), triggering monoubiquitination of FANCD2 and FANCI, which initiates repair of ICL-DNA.

FANCD2 and FANCI form a complex and are mutually dependent on one another for their respective monoubiquitination. After DNA damage and during S phase, FANCD2 localizes to discrete nuclear foci that colocalize with proteins involved in homologous recombination repair, such as BRCA1 and RAD51. The FA pathway is regulated by ubiquitination and phosphorylation of FANCD2 and FANCI. ATR-dependent phosphorylation of FANCI and FANCD2 promotes monoubiquitination of FANCD2, stimulating the FA pathway (Cohn and D'Andrea 2008, Wang 2007). The complex of USP1 and WDR48 (UAF1) is responsible for deubiquitination of FANCD2 and negatively regulates the FA pathway (Cohn et al. 2007).

Monoubiquitinated FANCD2 recruits DNA nucleases, including SLX4 (FANCP) and FAN1, which unhook the ICL from one of the two covalently linked DNA strands. The DNA polymerase nu (POLN) performs translesion DNA synthesis using the DNA strand with unhooked ICL as a template, thereby bypassing the unhooked ICL. The unhooked ICL is subsequently removed from the DNA via nucleotide excision repair (NER). Incision of the stalled replication fork during the unhooking step generates a double strand break (DSB). The DSB is repaired via homologous recombination repair (HRR) and involves the FA genes BRCA2 (FANCD1), PALB2 (FANCN) and BRIP1 (FANCJ) (reviewed by Deans and West 2011, Kottemann and Smogorzewska 2013). Homozygous mutations in BRCA2, PALB2 or BRIP1 result in Fanconi anemia, while heterozygous mutations in these genes predispose carriers to primarily breast and ovarian cancer. Well established functions of BRCA2, PALB2 and BRIP1 in DNA repair are BRCA1 dependent, but it is not yet clear whether there are additional roles for these proteins in the Fanconi anemia pathway that do not rely on BRCA1 (Evans and Longo 2014, Jiang and Greenberg 2015). Heterozygous BRCA1 mutations predispose carriers to breast and ovarian cancer with high penetrance. Complete loss of BRCA1 function is embryonic lethal. It has only recently been reported that a partial germline loss of BRCA1 function via mutations that diminish protein binding ability of the BRCT domain of BRCA1 result in a FA-like syndrome. BRCA1 has therefore been designated as the FANCS gene (Jiang and Greenberg 2015).

The FA pathway is involved in repairing DNA ICLs that arise by exposure to endogenous mutagens produced as by-products of normal cellular metabolism, such as aldehyde containing compounds. Disruption of the aldehyde dehydrogenase gene ALDH2 in FANCD2 deficient mice leads to severe developmental defects, early lethality and predisposition to leukemia. In addition to this, the double knockout mice are exceptionally sensitive to ethanol consumption, as ethanol metabolism results in accumulated levels of aldehydes (Langevin et al. 2011).