The long term goals of this proposal are to understand the fundamental mechanisms of eukaryotic chromosomal DNA replication and to determine how these mechanisms are disrupted in transformed cells. Transformation of cells involves deregulation of the initiation of chromosomal DNA replication. Therefore, their research will potentially give information concerning the steps of eukaryotic DNA replication that act as regulatory control points inside the cell. They will use simian virus 40 (SV40) as a model system in vitro to explore the mechanism of eukaryotic DNA replication. First, they will isolate and characterize human proteins that physically interact with human replication protein A (hRPA). Second, they have found that the DNA helicase activity of the SV40 large tumor antigen (T antigen) is 15-fold greater when T antigen binds DNA as a double hexamer rather than as a hexamer. The interaction of the T antigen double hexamer with synthetic replication bubble substrates (ie., substrates that contain a central ssDNA region flanked by duplex DNA) will be probed by chemical and enzymatic reagents. They will examine the role of the T antigen double hexamer on unwinding of lengthy DNA molecules, and determine the effect of other replication components on the stability of the T antigen double hexamer. Third, they will characterize the mechanism of DNA unwinding for the T antigen DNA helicase. They will determine the DNA helicase step size and the DNA contacts that T antigen makes during DNA unwinding through the use of ribose-substituted DNA forks. The model that T antigen encircles one of the two single-strands at a replication fork will be tested. Fourth, they will examine the DNA unwinding reaction mediated by hRPA on pseudo-origin substrates. They found previously that hRPA can bind and denature DNA substrates containing a structural feature (an 8 nt bubble) found within the ATP-dependent T antigen-SV40 origin complex. They will examine the role of pseudo-origin sequence and structure on hRPA binding and unwinding of these substrates.
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