The ability to modify genes in living cells by homologous recombination has significance to gene replacement therapy as well as site specific gene modification. During the past few years it has been demonstrated that mammalian somatic cells have all the enzymes necessary to mediate homologous recombination between exogenously introduced plasmids. The mechanism of this process and the actual nature of enzymes mediating the reactions are not known. As a first step towards a detailed genetic, biochemical and molecular biological understanding of mammalian honologous recombination, we have developed a cell-free system which is capable of catalyzing this reaction. The assay involves co-incubation of two non reverting, non-overlapping deletion mutants of PSV2neo, a eukaryotic-prokaryotic shuttle vector with the cell extracts and using the resulting DNA for direct analysis or transformation of recombination deficient (recA-) E-coli. The current proposal involves a detailed analysis of the cell extracts. The PSV2neo plasmids will be modified so that the two plasmids will have different restriction enzyme sites. THese restriction enzyme site polymorphisms will be used to examine the products of recombination and deduce the role of gene conversion and reciprocal recombination. The cell extracts will be tested for the presence of enzymatic activities that are required for the initiation and completion of the recombination reaction. Both biological and biochemical assays will be used. Isolation of a recombinase protein which would be involved in a DNA strand exchange reaction by fractionation methods and by antibody affinity columns is also proposed. A highly specific antibody will also be used to screen a human cDNA library in a bacteriophage expression vector to isolate the recombinase cDNA. The cDNA will be inserted into mammalian expression vectors and its ability to enhance homologous recombination will be studies. Isolation of a genomic clone corresponding to the cDNA and its characterization are also proposed.
