The study of homologous and nonhomologous recombination in animal cells is in its infancy. For the near future, the genetic analysis of these processes will involve construction of increasingly sophisticated DNA substrates, which can be introduced into cells to probe in detail their recombinational capabilities. Transfection of modified SV40 genomes into monkey cells offers an especially powerful approach to these studies 1) because the input genomes can be manipulated to distinguish among potential mechanisms, and 2) because the products of recombination are confined within the same plaque, allowing examination of the DNA segments that interacted in the exchange. This latter advantage is unique to the SV40 system. Experiments in this system and others have begun to outline the mechanisms of homologous and nonhomologous recombination in animal cells. In this application we propose to extend our characterization of these processes in monkey cells. Our experiments focus on specific mechanistic steps that are amenable to attack using the SV40 system; in several cases they will provide critical tests of the models we and others have put forward. Our specific objectives are summarized under the more general questions listed below. 1. Are regions of single-stranded DNA involved in the breakage and joining steps of nonhomologous recombination? 2. Are mismatches in heteroduplex intermediates in homologous recombination repaired efficiently and can the enzymatic activities be isolated? 3. Are both potential products or only one preserved in individual homologous exchange? 4. What is the basis for the competition between homologous and nonhomologous recombination and can it be controlled?
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