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Visualization of genetic recombination intermediates of human adenovirus type 2 DNA from infected HeLa cells
Abstract:
The study of recombination in prokaryotes has been facilitated by the availability of recombinational mutants and simple genetic elements such as phages and plasmids. These small but defined molecules of DNA have been especially useful for electron microscopic analysis of structural detail of molecules undergoing recombination both in vivo and in vitro. A limitation in the structural analysis of plasmid recombination is the absolute number of recombining molecules which can be identified and analysed amidst a background of nonrecombining molecules. This limitation would be of even greater consequence in studies of genetic recombination in animal cells. We therefore chose virus-infected animal cells as a model system for the study of the molecular mechanism of genetic recombination in higher organisms. HeLa cells infected with adenovirus serotype 2 (Ad-2) offer several advantages for studying recombination: (1) the virus contains a small and well characterized genome of about 35 kilobases; (2) a large amount of Ad-2 DNA is accumulated during lytic infection and host DNA synthesis is suppressed; (3) Ad-2 recombines at a very high frequency; and (4) similar to phages, animal viruses and Ad-2 in particular are believed to use many of the host cell's enzymes in necessary metabolic processes, presumably including recombination. In this study we used electron microscopic techniques to visualize the structures of in vivo Ad-2 DNA recombination intermediates. Molecules were observed with structures at putative cross-over points which were consistent with the molecular mechanism of recombination proposed by Holliday. In addition, we observed Ad-2 DNA molecules engaged in recombination which were simultaneously serving as templates for replication and/or transcription. To the best of our knowledge, this is the first visualization of in vivo recombination intermediates of discrete DNA molecules isolated from eukaryotic cells.