MECHANISM FOR GENETIC RECOMBINATION IN MAMMALIAN CELLS

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Mitotically dividing mammalian cells posses the recombinational machinery to catalyze a number of different types of DNA rearrangements that can alter either the expression of genes or their spatial configuration. Until recently, these events have been difficult to analyze because suitable genetic and model systems have not been available to probe these multiple recombinational activities. As a consequence, very little is known about the mechanisms or enzymology of recombination, even though these processes are likely to be important for the proper functioning of most cells. Our approach to this problem has been to use simple model systems, based on recombination substrates constructed in vitro to analyze different recombinatin events. These substrates are eukaryotic expression vectors containing either truncated or mutant copies of dominant selectable marker genes, such that reciprocal recombination or gene conversion restores a functiinal copy of the gene, following transient infection or stable integration of these substrate DNAs into appropriate cells. We are interested in studying the frequencies and mechanisms of extrachromosomal, intrachromosomal and interchromosomal, reciprocal and non-reciprocal recombination. Because extrachromosomal recombination of newly transfected DNA appears to be much more frequent than chromosomal recombination, we wish to determine the basis for these different frequencies. Gap repair and its possible applicatious will be explored. Among our long term interests are the exchange of genetic informatin between the chromosome and extrachromosomal plasmids, as well as the enzymology of recombination. We therefore, plan to explore methods for gene targeting ad gene recovery and hope to develop an in vitro system to study recombination. Many of the assasy for reciprocal and non-reciprocal events will also be extended to human cells including those that are believed to be aberrant in recombination and repair, and the effect of drugs and DNA damaging agents will be evaluated. We hope that the study of these basic processes will contribute to a better understanding of how recombination occurs and how it might be modulated in mammalian cells.