Fanconi anaemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Eight genes have been described that are mutated to cause FA; but many patients are not mutated in any of them; and the mechanism underlying the FA pathway remains unclear, because most FA proteins lack recognizable structural features or any identifiable biochemical activity. Recent evidence suggests that FA proteins function in a DNA damage response pathway involving the proteins produced by the breast cancer susceptibility genes BRCA1 and BRCA2. A key step in that pathway is a modification of an FA protein, FANCD2. The modification, monoubiquitylation, results in redistribution of FANCD2 to specific spots in the nucleus where BRACA1 also localizes. Five other FA proteins (FANCA, -C, -E, -F, and -G) have been found to interact with each other to form a multiprotein nuclear complex, the ?FA core complex?. This complex functions upstream in the pathway and is required for FANCD2 monoubiquitylation. However, none of the FA proteins contain an ubiquitin ligase motif or activity, and little is known about the ubiquitylation mechanism. We have purified the FA protein core complex and found that it contains four new components in addition to the five known FA proteins. One new component of this complex, termed PHF9, possesses ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitylation in vivo. PHF9 is defective in a cell line derived from a Fanconi anemia patient, and therefore represents a novel Fanconi anemia gene (FANCL). Our data suggest that PHF9 plays a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for FANCD2 monoubiquitylation. This work has now been accepted for publication in a leading journal, Nature Genetics Our work identifies the first Fanconi anemia gene that encodes a product with a catalytic activity. The discovery of PHF9/FANCL might provide a potential target for new therapeutic modalities. Over 95% of all FA patients are defective in FANCD2 monoubiquitylation. Thus, for example, a mutant version of PHF9 capable of ubiquitylating FANCD2 in the absence of other FA proteins, or a drug that could activate it, might provide a tool to mitigate disease symptoms in these patients. Conversely, specific inhibitors of the ubiquitin ligase activity might be used to sensitize tumor cells to the cytostatic effect of DNA cross-linking agents such as cisplatinum. More generally, the Fanconi pathway is involved in the DNA repair mechanisms that are often implicated as disrupted in cancer and possibly in aging. Now, we are investigating whether three other components of the FA core complex are also novel FA genes. Preliminary data suggest that one of them is. Moreover, one of the new component has recognizable enzymatic domains. We are investigating whether these domains are required for the function of the FA core complex. In summary, we believe that this project will not only lead to discovery of novel FA genes, but also shed light into the mechanism of the FA pathway.
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