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 chromo somal breakage, birth defects, and susceptibility to bone marrow failure an d cancer. At least seven complementation groups have been identified, and t he genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene prod ucts defective in FA cells are not completely understood, a clue to the fun ction 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 peroxid ases. We evaluated this hypothesis by mutagenesis and functional complement ation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on kno wn biochemical and cellular functions of FANCA. While the substitution muta nts were comparable to wildtype FANCA with regard to their stability, subce llular localization, and interaction with FANCG, only the Trp(183)-to-Ala s ubstitution (W183A) abolished the ability of FANCA to complement the sensit ivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apo ptosis after exposure to H2O2 showed no differences between parental FA gro up A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis:for th e expression of the peroxide-sensitive heme oxygenase gene showed appropria te induction after H2O2 exposure. Thus, W183A appears to be essential for t he in vivo activity of FANCA in a manner independent of its interaction wit h FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears t o alter the peroxide-induced apoptosis or peroxide-sensing ability of FA gr oup A cells. (C) 2001 Academic Press.