MECHANISM OF DNA REPLICATION IN EUCARYOTES--II SV40 AS A MODEL SYSTEM

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DNA topoisomerases I and II, DNA primase, DNA-dependent ATPase and RNase H activities, all of which are expected to participate in DNA replication, have been purified to near homogeneity from human cell lines and calf thymus and monoclonal and polyclonal antibodies have been raised against each proteins. To determine whether these proteins are required for eucaryotic DNA replication, the soluble in vitro replication system of SV40 DNA (which mimics the in vivo system) has been established and used to test whether the antibodies inhibit in vitro SV40 DNA replication. This system makes possible not only the identification and purification of various DNA replication proteins but also the identification of other factors which maintain high fidelity of DNA synthesis. For measuring the fidelity of the in vitro SV40 replication system, chimeric M13mp2 phages have been constructed which consist of an SV40 DNA replication origin and the E. coli lacZ gene.