Rabbit Anti-Human FANCL Polyclonal antibody

Fanconi Anemia (FA) is a rare genetic disease characterized by chromosome instability mostly resulting from an improper regulation of FANCD2 monoubiquitination. The E2 ubiquitin-conjugating enzyme UBE2T along with a multi-protein E3 ubiquitin-ligase complex containing a catalytic subunit FANCL mediates monoubiquitination of FANCD2. However, the upstream events involved in regulating FANCD2 monoubiquitination remain unclear. Here we report that HHR6, human homologs of the yeast ubiquitin-conjugating enzyme Rad6, regulates FANCD2 monoubiquitination in a manner distinct from that of UBE2T. Indeed, although downregulation of HHR6 compromised FANCD2 monoubiquitination and overexpression of HHR6 enhanced FANCD2 monoubiquitination, HHR6 did not directly interact with FANCL. Cells deficient in HHR6, UBE2T or FANCL did, however, all exhibit similar sensitivities to the DNA crosslinking agent mitomycin C (MMC). As an HHR6-induced increase in oncogenic potential could be partially suppressed by co-expression of non-monoubiquitinated FANCD2, a tight regulation of appropriate levels of monoubiquitinated FANCD2 appears to play an important role in tumor suppression. Thus, these results provide further insights into the regulation of FANCD2 monoubiquitination as well as indicate a common link between the FA-BRCA and HHR6 pathways in the maintenance of genome integrity.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.