Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis

Fanconi anemia is a chromosomal breakage disorder with eight complementatio n groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identif ied. Initial investigations of the interaction between FANCA and FANCC, pri ncipally by coimmunoprecipitation, have proved controversial. We used the y east two-hybrid assay to test for interactions of the FANCA, FANCC, and FAN CG; proteins. No activation of the reporter gene was observed in yeast coex pressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC. However, a high level of activation was foun d when FANCA was co-expressed with FANCG, indicating strong, direct interac tion between these proteins. Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dim erisation. The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG. Although both N- and C-terminal f ragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper inte raction with FANCA. (C) 2000 Academic Press.