The role of the figure 8 configuration of DNA as an intermediate in genetic recombination in the single-stranded DNA phage is being investigated with the aim of providing a description of a genetic system in which considerations of mechanism are based on the participation of an isolable intermediate of known properties. Two lines of research are proposed: (1) Branch migration is the movement of DNA strands through the junction of the figure 8 in such a way as to exchange strands and thus genetic information between two participating phage genomes with the formation of heteroduplex DNA. Measurements of the kinetics of branch migration that are important for assessing its role in vivo as the central feature of an intermediate in genetic recombination, the Holliday structure. These will include its rate, the possiblity of the modification of the rate by small molecules and protein components of the cell, the effect of known mismatches on the rate and the effect of superhelicity. (2) The study of genetic recombination in the single-stranded DNA phages by the single-burst method permits the identification of the products of a single genetic event and provides evidence for mechanism in a way that cannot be done by mixed burst analysis. A series of single-burst experiments has been completed that support the position of the figure 8 as an intermediate. Extensions are planned to the analysis of single bursts in spheroplasts. This will allow the testing of monomers, circular dimers, catenanes and figure 8s that are genetically labeled to give both heteroduplex and heterozygous species. These species can be constructed by modern methods of manipulating DNA. Particular problems that will be addressed are the mode of resolution of the figure 8, the possible participation of the circular dimer in recombination and certain aspects of mismatch repair that were suggested by our single-burst experiments in cells.
| Mulrooney, S B; Fishel, R A; Hejna, J A et al. (1996) Preparation of figure 8 and cruciform DNAs and their use in studies of the kinetics of branch migration. J Biol Chem 271:9648-59 |
| Camien, M N; Warner, R C (1986) Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects. J Biol Chem 261:6026-33 |
| Fishel, R A; Warner, R C (1986) Novel dimeric configurations from bacteriophage G4 replicative form DNA. Virology 148:198-209 |
