Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways

Abstract

Fanconi anaemia (FA) is a chromosomal instability disorder characterized by cellular sensitivity to DNA interstrand crosslinking agents and a high risk of cancer. Six of the eight proteins encoded by the known FA genes form a nuclear complex which is required for the monoubiquitination of the FANCD2 protein. FANCD2 complexes and colocalizes with BRCA1, but its presumptive role in DNA repair has not yet been clearly defined. We used yeast two-hybrid analysis to test for interaction between FANCD2 and 10 proteins involved in homologous recombination repair. FANCD2 did not interact with RAD51, the five RAD51 paralogs, RAD52, RAD54 or DMC1. However, it bound to a highly conserved C-terminal site in BRCA2 that also binds FANCG/XRCC9. FANCD2 and BRCA2 can be coimmunoprecipitated from cell extracts of both human and Chinese hamster wild-type cells, thus confirming that the interaction occurs in vivo. Formation of nuclear foci of FANCD2 was normal in the BRCA2 mutant CAPAN-1 cells, which indicates that the recruitment of FANCD2 to sites of DNA-repair is independent of wild-type BRCA2 function. FANCD2 colocalized with RAD51 in foci following treatment with mitomycin C or hydroxyurea, and colocalized very tightly with PCNA after treatment with hydroxyurea. These findings suggest that FANCD2 may have a role in the cellular response to stalled replication forks or in the repair of replication-associated double-strand breaks, irrespective of the type of primary DNA lesion.

Regulation of monoubiquitinated PCNA by DUB autocleavage

“…However, it is possible that USP1 may regulate the levels of other monoubiquitinated substrates in DNA repair pathways. Recent studies by our group and others have found that FANCD2 and PCNA colocalize at DNA repair foci during certain conditions of replication stress 17,18 (and data not shown). Therefore, we explored whether USP1 can deubiquitinate PCNA -a monoubiquitinated protein that localizes and functions at the DNA replication fork after UV damage or replication arrest.…”

The Fanconi Anemia/BRCA pathway: new faces in the crowd

“…Complex 1 is also essential for the inducible monoubiquitination of FANCD2 following cellular exposure to DNA damaging agents, such as MMC, ultraviolet (UV), or ionizing radiation (IR) and deoxynucleotide depletion due to hydroxyurea (Garcia-Higuera et al 2001;Gregory et al 2003). Following monoubiquitination, FANCD2 relocalizes from a soluble nuclear compartment to the chromatin fraction (Wang et al 2004) where it colocalizes in nuclear foci with other DNA damage response proteins, including FANCD1/BRCA2 (Hussain et al 2004;Wang et al 2004) BRCA1 (Garcia-Higuera et al 2001), RAD51 (Taniguchi et al 2002a;Hussain et al 2004), Proliferating Cell Nuclear Antigen (PCNA) (Hussain et al 2004), and Nijmegen Breakage Syndrome (NBS1) (Nakanishi et al 2002). Following relocalization to chromatin, monoubiquinated FANCD2 (FANCD2-Ub) promotes chromatin loading of the FANCD1/BRCA2 protein, forming a second complex (complex 2), where it may facilitate the formation of nuclear foci containing the homologous recombination (HR) DNA repair protein RAD51 (Wang et al 2004).…”

“…Accumulating evidence suggests a major function of complex 1 is to generate monoubiquitinated FANCD2 (FANCD2-Ub), required for the assembly of the FANCD2-Ub/BRCA2 chromatin complex (complex 2). Following monoubiquitination, FANCD2 relocalizes to DNA damage-induced S-phase foci where it associates with the DNA repair proteins BRCA2/FANCD1 (Wang (Nakanishi et al 2002), RAD51 (Hussain et al 2004), and PCNA (Howlett et al 2005). The formation of these DNA damage-induced FANCD2 foci is dependent on the presence of functional BRCA1 (Garcia-Higuera et al 2001), thereby linking this DNA repair protein to the FA pathway.…”

FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

“…FANCD2S+/FANCD2L+ phenotype in patients with a positive chromosome breakage test and FA clinical features suggests deleterious mutation on any of the genes functioning downstream of FANCD2 monoubiquitination. Three complementation groups corresponding to downstream proteins, that play their role after FANCD2 activation, have been identified (FANCD1/ BRCA2, FANCJ and FANCN) (9,15,19,28). Western blots for BRCA2 were performed on FANCD2S+/FANCD2L+ patients and all of them showed normal expression of this protein.…”

“…The activation of this complex induced by DNA replication or by DNA-damage results in monoubiquitination of FANCD2, which moves into areas of damaged chromatin to interact with downstream FA members (FANCD1/BRCA2, FANCJ, FANCN) and other DNA-repair proteins such as BRCA1 and RAD51. FANCD2 is the central protein that connects the FA/BRCA pathway to the homologous recombination-mediated DNA repair (18,19,23,(25)(26)(27)(28). The recently identified FANCI protein associates with FANCD2 and is the second monoubiquitinated component of the FA pathway (14,29).…”