Postreplication repair (or recombinational repair) is thought to be an important mechanism in mammalian cells and in microorganisms for overcoming DNA damages caused by radiations and chemical carcinogens. Little is known of the mechanism of this repair process in animal cells, but it has been investigated in some detail in E. coli. In this organism recombinational repair depends upon recA protein, product of the recA gene, which has been shown to promote homologous pairing and strand exchange reactions in vitro. Recently we have succeeded in identifying and partially purifying the yeast endonuclease that acts on Holliday crossed strand exchanges, an enzyme required for a late step in recombinational repair. We plan to extend our studies on the preparation and purification of this enzyme from yeast, and to investigate optimal conditions and to explore its action in cutting various substrates, supercoiled plasmids carrying inverted repeats, figure 8 or alpha shaped DNA molecules, which will be constructed by means of recA mediated reactions. We will also use stable crossed strand exchanges produced by annealing appropriate DNA molecules. We hope to determine the exact positions of the cuts by DNA sequencing methods. We also plan to look for a similar enzyme in extracts from E. coli. This crossed strand endonuclease will be tested in reactions with recA protein in an attempt to perform postreplication repair and genetic recombination in vitro.
