The objective is to gain an understanding of the control mechanisms operative in the regulation of eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A as the model system. The gene product of early region 2A is a 72,000 dalton single-stranded DNA-binding protein (DBP) that is involved in regulating viral gene expression in at least four ways: (1) the initiation and elongation steps of DNA synthesis, (2) the regulation of transcription of several of the Ad5 early genes, (3) stability of early mRNAs, and (4) the host-range of the virus. The immediate goal is to precisely define within the DBP domains that are involved in each of the functions of the protein. To accomplish this, we will generate mutations in the DBP by site-specific mutagenesis techniques and analyze the mutant DBPs for their ability to bind to single-stranded DNA, and to help adeno-associated virus replicate in human cells. In-depth probing of the functional properties of the protein will be performed following re-introduction of the mutated gene back into the virus.
