An in vitro system has been developed that synthesizes DNA semi- conservatively and the products are genetically active. A large form of DNA polymerase III has been isolated and the nature of the multimer components will be analyzed. A DNA ts mutant which is defective in a cofactor, but not in Pol3, has been found. The thermosensitive cofactor needs to be purified and its relation to replication complex will be analyzed. Using the above in vitro system, complementation analysis of all 9 gene functions has been performed. Four more cofactors need to be purified and studied. Phage SPP-l uses the host polymerase III for its replication of DNA. The polymerase III is modified by the product of one functional unit with two complementing polypeptides that expresses very early after infection. The nature of this modification, the specificity of large Pol3 form, for any particular strands of phage, and the role of RNA initiators in DNA replication will be studied. The phage uses two other host functions, which are common to both host and phage DNA replication. These cofactors are currently under study. In both cases, the DNA products could be assayed by transformation and transfection. Recombination deficient mutants of one class accumulate non-covalently bonded donor and recipient DNA complexes. These will be isolated and used as a substrate for the development of an in vitro recombination system.