Fanconi anaemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Twelve 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 five 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. Much of this work has been published in Nature Genetics in 2003. The 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. [unreadable] [unreadable] In a paper published in Nature Genetics in 2004, we showed that the 95 kd subunit of the Fanconi anemia core complex is defective in FA complementation group B patients (the gene is named FANCB). The significance of this study can be summarized as follows. First, our study identifies the true FANCB gene that had eluded identification for more than 10 years. Before our paper, the identity of the FANCB gene was controversial, and has been suggested to be BRCA2. Our paper settled this issue for the field. Second, we find that FANCB is localized on the X-chromosome and subject to X-inactivation. This finding has changed the prevalent view that Fanconi anemia is a uniquely autosomally-inherited disease. Our paper thus has important clinical implication for diagnosis and genetic counseling for FA families. For example, the female carriers of FANCB mutation will have 50 percent of risk to conceive an affected son or a carrier daughter. These carriers should be identified and given proper counseling for risk. Third, all other genes that maintain genome stability are localized on autosomes and present in two copies. In contrast, FANCB is X-linked and present in only one active copy. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability, because it will only take one mutation to inactivate FANCB, but it will take two mutations to inactivate other genes. Our study suggests that FANCB may be mutated in cancer patients who do not have Fanconi anemia.[unreadable] In a paper published in Nature Genetics in 2005, we demonstrated that FAAP250 is mutated in FA patients of a new complementation group, FA-M. The gene encoding the FAAP250 protein was renamed FANCM. The importance of the FANCM findings is that although FA proteins have previously been implicated in DNA repair, the interactions between FA proteins and DNA are poorly understood, because the known FA proteins lack DNA-related enzymatic domains or activities. The newly discovered FANCM has a conserved helicase domain and a DNA-translocase activity. A companion paper in Nature Genetics identified another FA protein, FANCJ, as BACH1/BRIP1, a known DNA helicase. The discovery of two FA proteins with helicase domains or activities suggests a mechanism of direct participation in DNA repair by the FA proteins. Our data suggest that FANCM may have at least three important roles in the FA DNA damage response pathway. First, FANCM may have a structural role to allow assembly of the FA core complex, because in its absence, the nuclear localization and stability of several FA proteins are defective. Second, FANCM may act as an engine that translocates the core complex along DNA. Speculatively, this translocation may allow the core complex to sense and locate to the damaged DNA, which could be an important step either before or after FANCD2 monoubiquitination. Third, FANCM is hyperphosphorylated in response to DNA damage, suggesting that it may serve as a signal transducer through which the activity of the core complex is regulated. A DNA damage checkpoint kinase, ATR, has been shown to act upstream of the FA pathway, but its substrate has not been defined. FANCM contains multiple predicted ATR phosphorylation sites, and may serve as a substrate through which ATR regulates FANCD2 monoubiquitination.[unreadable] [unreadable] We have since identified another component of the FA core complex, FAAP100, and shown that this protein is required for stability and a key function of the complex--FANCD2 monoubiquitination. Thus, all nine components of the core complex are essential for this ubiquitination reaction, suggesting that the entire complex is a machine that works concertedly to monoubiquitinate FANCD2. We also found that deficiency in FAAP100 generated by siRNA depletion or gene knockout results in cellular phenotypes that are hallmark features of FA cells. Therefore, FAAP100 should be an essential component of the FA-BRCA network, and its defects in human should also cause FA.[unreadable] [unreadable] More recently, we have identified a new component of the FA core complex, termed HIP. We find that HIP can recognize structured DNA that mimics intermediates generated during DNA replication. We are now using siRNA depletion and gene knockout method to examine the function of HIP.