Intracellular localization of the Fanconi anemia complementation group A protein.
Mutations in the Fanconi anemia (FA) complementation group A (FANCA) gene leads to bone marrow failure, developmental abnormalities and cancer predisposition. To map the intracellular site of FANCA, we constructed a plasmid vector which linked in-frame the enhanced green fluorescent protein (EGFP cDNA) to the 5' end of the FANCA cDNA (pDAS-3). We studied the expression of pDAS-3 in the FANCA mutant fibroblast cell line (GM6914). MMC sensitivity of pDAS-3 transfected cells was comparable to wild-type fibroblasts. The resulting fluorescence pattern in the stable pDAS-3 cell line expressing the fusion protein was primarily nuclear. EGFP-selected cells (lacking FANCA) remain hypersensitive to MMC and maintained a cytoplasmic fluorescence pattern. Using deletion mutants of pDAS-3, a nuclear localization domain was identified at the amino terminus of the polypeptide. Western blot results of FANCA protein confirmed the presence of FANCA in nuclear fractions and FANCA protein levels did not vary during cell cycling. This nuclear trafficking of FANCA should guide future work in defining the function of this protein.
Walsh
C E
CE
UNC Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, 27599, USA. cwalsh@med.unc.edu
Yountz
M R
MR
Simpson
D A
DA
D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D051856:Fanconi Anemia Complementation Group Proteins; D005347:Fibroblasts; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology; D016296:Mutagenesis; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010641:Phenotype; D011506:Proteins / Q000032:analysis*; D011993:Recombinant Fusion Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051856:Fanconi Anemia Complementation Group Proteins; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D016685:Mitomycin