Our continued long-term objective is to gain an understanding of DNA replication and post-replicative modification, i.e., methylation of DNA, at the molecular level. Frog virus 3 (FV3) replication in eukaryotic cells is used as a model system to meet these objectives. The basic features of FV3 DNA replication are similar to eukaryotic DNA replication, i.e., DNA replication occurs at a fixed site on a linear molecule and the replication fork moves bidirectionally. However, the FV3 genome replicates in two physically separated stages, the first involving origin-specific DNA replication in the nucleus and the second involving recombination/replication in the cytoplasm. It may, therefore, be possible to unambiguously characterize and selectively purify DNA structures and proteins involved in origin-specific DNA replication or in recombinant/replication. We will develop an origin-specific, in vitro FV3 DNA replication system and using this system, determine the minimal functional origin of the FV3 genome. Deletion and site-directed mutants of the minimal origin will be utilized in the in vitro system to define the sequence specificity and structural features required to initiate FV3 DNA replication. Drug-resistant and temperature-sensitive mutants of FV3 will be utilized to analyze the structure (nucleotide sequence) and function of genes that are involved in the initiation, chain elongation and termination of DNA replication. FV3 DNA is heavily methylated by a virus-specified DNA methyltransferase. One function of the DNA methylation is that it protects the viral genome from the action of a viral-specific endonuclease. Our genetic analysis of DNA methyltransferase and endonuclease indicates that these two enzymes may constitute a restriction-modification (R-M) system. We will purify and biochemically characterize those two enzymes and isolate and analyze their genes to establish whether or not FV3 DNA methyltransferase and endonuclease constitute an R-M system.
