The long term objective of this research project is to gain an understanding of DNA replication and post-replicative modification, i.e., methylation of DNA at the molecular level. Frog virus 3 (Fv 3) replication in eukaryotic cells is used as a model system to meet these objectives. FV 3 DNA replicates in two stages. During the first stage, genome or less than genome length molecules are synthesized in the nucleus only. By contrast, in the second stage, viral DNA exclusively replicates as concatemers in the cytoplasm. It is proposed that concatemeric DNA replication represents chain elongation, whereas DNA replication in the first stage is initiated at the origin(s) of DNA replication. The origin(s) of FV 3 DNA replication will be determined by restriction endonuclease analysis, by electron microscopy, and by the ability of individual restriction fragments to replicate after transfection into the cells. We will then determine whether or not the origin(s) of FV 3 DNA replication is used only in the first stage (nuclear) of FV 3 DNA replication. The mechanism of concatemer formation will be determined by using two species of defective interfering particles of FV 3 having differential sensitivity to restriction endonuclease Eco RI. Several temperature-sensitive mutants of FV 3, defective in second stage DNA replication, will be isolated and characterized. FV 3 DNA is heavily methylated, probably by a virus-specific methylase. Preliminary indications are that methylation of FV 3 DNA protects it from the action of a virus-specific endonuclease. Virus-specificity of these two enzymes will be established (i) by genetic analysis, (ii) by marker rescue experiments, and (iii) by purification and biochemical characterization. We will also determine whether or not these two enzymes constitute a system analogous to bacterial restriction-modification systems.
