Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis.
Fanconi anemia is a chromosomal breakage disorder with eight complementation groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified. Initial investigations of the interaction between FANCA and FANCC, principally by co-immunoprecipitation, have proved controversial. We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins. No activation of the reporter gene was observed in yeast co-expressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC. However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins. Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dimerisation. The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG. Although both N- and C-terminal fragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper interaction with FANCA.
Huber
P A
PA
Division of Medical Genetics, Guy's, King's and St. Thomas' School of Medicine, Guy's Hospital, 7th Floor, Guy's Tower, London, SE1 9RT, United Kingdom. pia.huber@kcl.ac.uk
Medhurst
A L
AL
Youssoufian
H
H
Mathew
C G
CG
D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005199:Fanconi Anemia / Q000235:genetics* / 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; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D066298:In Vitro Techniques; D009687:Nuclear Proteins; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; 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; D009687:Nuclear Proteins; D011506:Proteins