Little is known about initiation of DNA replication in eukaryotic organisms or how these events are regulated. Studies of initiation of DNA replication and its regulation are important because these processes are believed to play central roles in diverse biological events, such as control of development and cancer. The Simian Virus 40 (SV40) in vitro replication system is an excellent model for studies of initiation of eukaryotic replication. Sensitive assays have been developed that can be used to study SV40 initiation events prior to DNA synthesis and to follow replication events seconds after DNA synthesis initiates. Equally important, it is now possible to examine the regulation of these events; a process that can theoretically occur at any stage of initiation. The experiments in this proposal are designed to establish the mechanism of initiation of SV40 DNA synthesis in vitro. They are also designed to further characterize the enzymatic requirements for initiation of SV40 DNA replication. Finally, the experiments in this proposal are intended to more fully describe aspects of the regulation of initiation of SV40 DNA synthesis in vitro. Initiation events prior to DNA synthesis will be followed using previously described unwinding assays. Pulse and pulse-chase reactions will be used to generate nascent DNA, including primer-DNA, formed seconds after the start of DNA synthesis. These DNAs will be used to establish the mechanism of initiation of DNA synthesis. Similar studies will define the enzymatic requirements for initiation of DNA synthesis. Insights into the dNTP dependent of the preinitiation complex in the vicinity of the SV40 origin will be obtained by determining the size and position of the unwound region in the form UR. The knowledge derived from studies of initiation of SV40 DNA replication in permissive and nonpermissive extracts should provide insights into initiation of chromosomal DNA replication.
