Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiquitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation-induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.

a molecular complex with primarily nuclear localization (Kupfer et al., 1997; Garcia-Higuera et al., 1999; Waisfisz et al., 1999a). FANCC also localizes to the cytoplasm,

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.

Activation of phosphatidylinositol 3Ј-kinase (PI 3-kinase) 1 appears to play a pivotal role in signal transduction by growth factors and insulin (1, 2). PI 3-kinase has the unusual property of phosphorylating phosphatidyinositides on the 3Ј position generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) in vivo (3). These two products are thought to act as second messengers in processes such as mitogenesis (4, 5), oncogenic transformation (6), apoptosis (7), and various types of membrane trafficking (8, 9). Specifically, PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 bind to the pleckstrin homology (PH) domains of the Akt/PKB protein kinase and of the PDK1 protein kinase which activates Akt/PKB (10 -15). Binding of PtdIns(3,4,5)P 3 activates PDK1 and PtdIns(3,4)P 2 has been reported to preferentially activate Akt/PKB (12-16). In addition, PtdIns(3,4)P 2 and PtdIns (3,4,5)P 3 activate several isoforms of protein kinase C (17), and in vitro binding of PtdIns(3,4,5)P 3 to the SH2 domains of p85 and Src SH2 has been demonstrated (18).The production of some forms of phosphoinositides depends on the activities of inositol polyphosphate-5-phosphatases. Many different species of these enzymes exist, with a recently described member, SHIP (for SH2 domain-containing inositol 5-phosphatase), being implicated in receptor signaling in hematopoietic cells) (19 -21). SHIP is tyrosine phosphorylated in response to treatment of cells with erythropoietin, interleukin-2, interleukin-3, macrophage colony-stimulating factor, B cell receptor cross-linking, and T cell activation (22); however, in most cases, phosphorylation does not appear to affect activity. SHIP has an SH2 domain, multiple proline-rich sites representing possible sites of interaction with SH3 domains, two NPXY motifs, and associates with Shc, Grb2, and SHP-2 under certain conditions (23-25). The 5Ј-phosphatase activity of SHIP is specific for phosphatidyinositols and inositol which are phosphorylated at the 3Ј position (23). In some systems, SHIP appears to negatively regulate cell growth (21) or induce apoptosis (25), perhaps counteracting growth factor signals. It is not clear whether tyrosine phosphorylation of SHIP promotes or diminishes these inhibitory effects.Since PI 3-kinase appears to play a fundamental role in signal transduction in all mammalian cell types and SHIP expression is limited to hematopoietic cells, we examined whether the more widely expressed SHIP-related protein 51C/ SHIP2 (26, 27) is involved in signaling from receptor tyrosine kinases in other cells. EXPERIMENTAL PROCEDURESReagents-Rabbit antisera were raised against a glutathione S-transferase fusion protein containing carboxyl-terminal residues 1105-1213 of the SHIP2 sequence (27) as described previously (28 Cell Culture-SH-SY5Y human neuroblastoma cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. PC12 cells (a kind gift from D. Maysinger, McGill University) were grown in Dulbecco's modif...

Background: Different mediators assist RecA to catalyze genetic recombination. Results: DprA facilitates the displacement of both SSBs (SsbB and SsbA), increases RecA nucleation onto SSB-coated ssDNA, and mediates DNA strand annealing. Conclusion: DprA facilitates RecA-mediated strand exchange and anneals complementary strands coated by an SSB protein.Significance: RecA-dependent chromosomal transformation and RecA-independent plasmid transformation require the competence-induced DprA mediator.