AN E2F DOMINANT-NEGATIVE MUTANT BLOCKS E1A INDUCED CELL-CYCLE PROGRESSION

E2F is a cellular transcription factor that is regulated during the ce ll cycle through interactions with the product of the retinoblastoma s usceptibility gene (RB1) and the pRb-like p107 and p130 proteins. Anal ysis of mutations within both adenovirus E1A and pRb, which affected t heir ability to regulate cellular proliferation and alter E2F activity , suggested that E2F may play a role in cell cycle progression. Microi njection of a GST-E2F-1 fusion protein into quiescent Balb/c 3T3 cells induced DNA synthesis whereas co-injection of GST-E2F-1 and GST-E2F(9 5-191) protein, encoding only the DNA binding domain of E2F-1, blocked the induction of S-phase. While E1A likely targets multiple cellular pathways, co-injection of the GST-E2F(95-191) dominant inhibitory prot ein with 12S E1A protein blocked E1A-mediated induction of DNA synthes is, suggesting that the E2F-dependent pathway is dominant. Analysis of the interval required for microinjected quiescent cells to enter S-ph ase indicated that E2F-1 acted faster than either E1A or serum.