FANCC, FANCE, and FANCD2 form a ternary complex essential to the integrity of the Fanconi anemia DNA damage response pathway.
Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, cancer predisposition, and increased cellular sensitivity to DNA-cross-linking agents. The products of seven of the nine identified FA genes participate in a protein complex required for monoubiquitination of the FANCD2 protein. Direct interaction of the FANCE protein with both fellow FA complex component FANCC and the downstream FANCD2 protein has been observed in the yeast two-hybrid system. Here, we demonstrate the ability of FANCE to mediate the interaction between FANCC and FANCD2 in the yeast three-hybrid system and confirm the FANCE-mediated association of FANCC with FANCD2 in human cells. A yeast two-hybrid system-based screen was devised to identify randomly mutagenized FANCE proteins capable of interaction with FANCC but not with FANCD2. Exogenous expression of these mutants in an FA-E cell line and subsequent evaluation of FANCD2 monoubiquitination and DNA cross-linker sensitivity indicated a critical role for the FANCE/FANCD2 interaction in maintaining FA pathway integrity. Three-hybrid experiments also demonstrated the ability of FANCE to mediate the interaction between FA core complex components FANCC and FANCF, indicating an additional role for FANCE in complex assembly. Thus, FANCE is shown to be a key mediator of protein interactions both in the architecture of the FA protein complex and in the connection of complex components to the putative downstream targets of complex activity.
Gordon
Susan M
SM
Program in Genetics and Genomic Biology, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. susan.gordon@utoronto.ca
Alon
Noa
N
Buchwald
Manuel
M
D002460:Cell Line; D002470:Cell Survival; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005199:Fanconi Anemia / Q000235:genetics* / Q000378:metabolism*; D052218:Fanconi Anemia Complementation Group C Protein / Q000502:physiology*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000502:physiology*; D052239:Fanconi Anemia Complementation Group E Protein / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D047468:Immunoprecipitation; D016685:Mitomycin / Q000494:pharmacology; D008954:Models, Biological; D008957:Models, Genetic; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D003432:Cross-Linking Reagents; C497651:FANCC protein, human; C499907:FANCE protein, human; D052218:Fanconi Anemia Complementation Group C Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D052239:Fanconi Anemia Complementation Group E Protein; D016685:Mitomycin