Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein.
Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells.
Ren
J
J
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Youssoufian
H
H
D000409:Alanine / Q000737:chemistry; D017209:Apoptosis; D015153:Blotting, Western; D002470:Cell Survival; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D052217:Fanconi Anemia Complementation Group A Protein; D005816:Genetic Complementation Test; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008214:Lymphocytes / Q000166:cytology; D016685:Mitomycin / Q000494:pharmacology; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009195:Peroxidase / Q000737:chemistry*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D004268:DNA-Binding Proteins; C498471:FANCA protein, human; D052217:Fanconi Anemia Complementation Group A Protein; D019384:Nucleic Acid Synthesis Inhibitors; D011506:Proteins; D016685:Mitomycin; D006861:Hydrogen Peroxide; D009195:Peroxidase; D000409:Alanine