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Recently, hydrogen peroxide (H₂O₂), and its free-radical product the hydroxyl radical (OH·), have been identified as major sources of DNA damage in living organisms. We examined DNA repair of hydrogen peroxide damage, using a standard bacteriophage T4 test system in which several different types of repair could be determined. Post-replication recombinational repair and denV-dependent excision repair had little or no effect on H₂O₂ damage. Also, an enzyme important in repair of H₂O₂-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H₂O₂ damage to the phage. However, multiplicity reactivation, a form of recombinational repair between multiply infecting phage genomes, was found to repair H₂O₂ damages efficiently. The RAD52 gene of Saccharomyces cerevisiae and genes 46 and 47 of bacteriophage T4 are essential for most recombination and recombinational repair in their respective organisms. The RAD52 gene was introduced into expression vectors which were used to transform E. coli. RAD52 expression was induced, and its ability to complement either gene 46 or gene 47 phage mutants was determined with respect to phage growth, recombination, and recombinational repair. RAD52 gene expression allowed growth of gene 46 and gene 47 mutants under otherwise restrictive conditions, as measured by plaque formation and burst size. The RAD52 gene also restored the ability of gene 46 and gene 47 mutants to undergo recombination of rII markers. Furthermore, the RAD52 gene restored recombinational repair after UV irradiation of gene 46 and gene 47 mutants. The published DNA sequence of RAD52 was compared with the published sequences of genes 46 and 47. Although overall homologies were only marginally significant, RAD52 and gene 46 had substantial sequence similarity over a limited region. These results indicate that the recombinational repair pathway found in phage T4 may be ubiquitous for DNA damage caused by endogenous exidative reactions. Furthermore, they indicated that an essential element of the recombination mechanism in both procaryotic viruses and eucaryotes arose from a common ancestor. Procaryotes and eucaryotes are thought to have diverged at least one billion years ago. Thus, recombination apparently arose early in evolution.

Productive infection of mouse cells with polyoma virus yields mainly two types of particles: Complete virions and empty capsids. Empty polyoma capsids have been shown to be capable of interacting with DNA, in vitro, to form what has been referred to as polyoma-like particles (PLP). The particles are stable in high concentrations of salt and contain DNA protected by the capsid against the action of pancreatic DNase. The development of PLP into a gene transfer vehicle is the subject of the investigations described in the present dissertation. The approach has been to first, characterize the process of PLP formation and second, determine whether the genetic information contained in a specific DNA fragment and assembled into PLP in vitro can be transferred to cells and subsequently be expressed. In terms of PLP characteristics, the experimental results described in this dissertation show that the DNA extracted from PLP is heterogeneous in size. It has a mean molecular weight of 1.2 x 10⁶ with a standard deviation of ±0.5 x 10⁶. In addition, analysis of PLP DNA with restriction endonucleases revealed that a specific primary sequence or higher order structure is not required for PLP formation. Either linear, circular or supercoiled polyoma DNA, as well as, single-stranded DNA, rRNA and the synthetic homopolymers poly(dA).poly(dT) and poly(dG).poly(dC) can be used for PLP formation. Transfer of genetic information by PLP has been accomplished by using a restriction fragment containing the transforming sequences of polyoma DNA as a model gene. This fragment of polyoma DNA, which consists of 1,831 base pairs (approximately 1.2 x 10⁶ daltons) and extends clockwise from the BclI site to the EcoRI site on the conventional polyoma map, causes the induction of the transformed phenotype in rat cells grown in culture. Infection of rat F111 cells by PLP, containing this DNA fragment, results in DNA-mediated oncogenic transformation of the cells as indicated by the formation of dense foci. This gene transfer activity of PLP is shown to be 50 to 150 times more efficient than the widely used calcium phosphate coprecipitation method of introducing DNA into mammalian cells.