The large tumor antigens (T Ags) encoded by simian virus 40 (SV40) and polyomavirus (Py) are required for replication of the viral genome in infected cells. In vitro systems capable of replicating either SV40 or Py origin-containing DNA, mediated by SV40 or Py T Ags, respectively, have been established. These have provided the opportunity to determine the activities of the T Ags during replication, to identify cellular DNA replication factors, and to understand the mechanisms that regulate eukaryotic DNA synthesis. It is proposed to study aspects of both SV40 and polyoma DNA replication in vitro. Py T Ag, which has not been studied extensively, is expected to provide interesting contrasts to the more well characterized SV40 T Ag. The DNA binding properties, helicase, and unwinding activities of Py T Ag will be studied, and domains of the Py protein involved in DNA binding and other replication-associated activities will be identified. The roles of SV40 T Ag in various stages of DNA replication will be studied using a purified conditional (temperature sensitive) mutant in order to assess the roles of T Ag during both DNA initiation and chain elongation. The function of enhancer sequences in Py DNA replication will be studied by attempting to establish a system in which Py RNA replication in vitro is dependent upon or affected by the presence of enhancer sequences. A simplified enhancer region will be constructed and tested for its ability to activate the Py origin in vivo and in vitro. Murine enzymes and factors that are required for replication of Py DNA will be purified and characterized with the aim of studying their role in host species specificity as well as their interaction with enhancer-binding factors.