The long term objectives of the proposed project are three-fold. First, new substitution, deletion, insertion, and point mutations in the human adenovirus specified DNA binding protein (DBP) will be used to provide additional supporting evidence or new refuting evidence for the myriad of purported functions of DBP (e.g., in early gene turn on and off, DNA replication, late gene turn on, viral assembly, and oncogenic transformation). Second, an extensive collection of DBP mutants, which is in the process of being made, will be utilized to obtain a better understanding of the interrelationships of the many DBP functions. Third, chemical, biochemical and cell biological studies will be continued in order to better understand the mechanism(s) DBP uses to carry out its diverse set of roles, particularly as they apply to gene expression. In the latter, special emphasis will be directed to (i) understanding DBP interaction with RNA, (ii) deciphering the mechanism(s) by which this protein kills cells and (iii) determining its role in early gene expression. To achieve the first objective, our complementing, DBP expressing human lines will be utilized to construct many new DBP mutants. Two new schemes for mutagenesis will be attempted. They should facilitate the rapid construction of large numbers of diverse mutants in other adenovirus genes as well as DBP.
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