The Fanconi anemia core complex forms four complexes of different sizes in different subcellular compartments.
Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA crosslinking agents such as mitomycin C. The molecular mechanism for the disease remains elusive, but at least 6 FA proteins are known to be part of what is termed the FA core complex. We used affinity pulldown of FLAG-FANCA to pull down the FA complex from whole-cell extracts. Mass spectroscopy detected previously reported FA-binding proteins, including FANCA, FANCC, FANCG, cdc2, and GRP94, thus validating the approach. We further describe a method of purification of the FA core complex in an effort to find novel complex components and biochemical activity to define the function of the complex. By using conventional chromatographic fractionation of subcellular preparations, we report: (i) the FA core complex exists in a cytoplasmic form at 500-600 kDa; (ii) a larger, 750-kDa cytoplasmic form is seen only at mitosis; (iii) a nuclear form achieves a size of 2 megaDaltons; and (iv) a distinct 1-megaDalton FA core complex exists bound to chromatin that contains phosphorylated FANCA after undergoing DNA damage. We are continuing our analysis using mass spectroscopy in an effort to characterize novel binding proteins. These data will help define the biochemical role of the FA core complex in normal cell physiology as well as in the development of the FA disease state.
Thomashevski
Andrei
A
Department of Microbiology, University of Virginia Health System, University of Virginia, Charlottesville, Virginia 22908, USA.
High
Anthony A
AA
Drozd
Mary
M
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Grant
Patrick A
PA
Kupfer
Gary M
GM
D016203:CDC2 Protein Kinase / Q000737:chemistry; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry; D005199:Fanconi Anemia / Q000378:metabolism*; D052217:Fanconi Anemia Complementation Group A Protein; D052218:Fanconi Anemia Complementation Group C Protein; D052241:Fanconi Anemia Complementation Group G Protein; D051856:Fanconi Anemia Complementation Group Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry; D008938:Mitosis; D008954:Models, Biological; D009687:Nuclear Proteins; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D013347:Subcellular Fractions
D018797:Cell Cycle Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C498471:FANCA protein, human; C497651:FANCC protein, human; C499909:FANCG protein, human; D052217:Fanconi Anemia Complementation Group A Protein; D052218:Fanconi Anemia Complementation Group C Protein; D052241:Fanconi Anemia Complementation Group G Protein; D051856:Fanconi Anemia Complementation Group Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011506:Proteins; C018551:glucose-regulated proteins; D004247:DNA; D016203:CDC2 Protein Kinase; D010744:Phosphoric Monoester Hydrolases