The overall objective of this proposal is to define the molecular mechanisms by which SV40 T antigen regulates viral DNA replication. T antigen carries out a number of important and related regulatory functions in the course of infection. Our goals are to understand the nature of these functions, to relate these functions to one another, and to determine how the structure of T antigen is organized to accomplish these functions. The activities on which we focus on this application are the binding of T antigen to the origin of replication, the denaturation of origin duplex DNA, the role of T antigen in guiding cellular replication proteins to the origin, the nicking of origin DNA, and the switch of T antigen from initiation to helicase functions in DNA replication. We think that each of these steps in the replication of SV40 may be accomplished by distinct functions of T antigen. This information may establish general principles that are applicable to other replicons. The initiation of DNA replication is one of the most perfectly regulated processes in eukaryotic cells and is crucial to the control of cellular growth. We have the following specific aims: 1. To characterize further the structure and function of the origin of replication. We will construct insertion mutations between domains that we have previously identified to probe the spatial relationships among the domains, and we will isolate mutations in T antigen that suppress base substitution mutations in T antigen-binding regions of the origin to identify protein-DNA contact regions. 2. To analyze T antigen-induced events at the origin of replication. We will use established DNase, DMS and KMn04 footprinting techniques. We will also focus on a newly discovered, T antigen-associated nuclease activity directed at the AT domain of the core origin and on the interactions of T antigen with cellular replication proteins. 3. To characterize the domain structure of T antigen. We will use a two-stage saturation mutagenesis of the """"""""zinc finger"""""""",tsA, origin binding, origin unwinding, nuclease-association, and helicase domains of T antigen and we will select for second-site mutations in T antigen or in the origin of replication that suppresses first-site mutations in each functional domain of T antigen.
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