The proposed research is aimed broadly to contribute to an understanding of DNA function by application of electron microscopic methodology (EM) in the areas of recombination, replication and virus structure. Details of RecA promoted strand exchange (SE) and the circumstances preventing or stimulating RecA action will be considered with particular reference to: (i) how RecF, RecR, RecO, RuvA, RuvB and SSB modulate the action of RecA in homologous recombination and recombinational repair by EM analysis of in vitro experiments designed to test specific models for this process; (ii) the existence of triple stranded structures during RecA-mediated SE; (iii) the actual changes that can be observed and analyzed by EM correlating with the two successive ATP-independent and ATP-dependent phases of SE; (iv) the positions along the RecA filament (random, at ends, in a wave) of the association-dissociations of RecA; (v) whether SSB is present within active RecA filaments; and (vi) the translocation ability of RuvB and mode of influence by other factors. In another project, control of initiation at the gamma R6K plasmid origin will be examined. Unwinding at the AT-rich region by pr protein and six hyperactive mutants and the effect of super helical tension and DnaA or Integration Host Factor will be studied applying methods of EM, S1 nuclease sensitivity and radioactive incorporation. This will require standardization and simplification of several features of the in vitro R6K system. With gold labeled antibodies and EM, individual specific proteins will be identified in initiation complexes at the gamma origin. A third project involves HIV-1 Integrase. An as yet unidentified donor-target recombinant structure produced by integrase (IN) activity isolated by non-ionic detergent from HIV-1 virions will be studied. The structure has been identified by gel mobility and may be a potential intermediate in the concerted reaction. Gold labeled HIV-1 IN will be used to help determine if the complex has a different structure under low and high salt assay conditions which might help explain the specific deletions that are found in integration products produced under low salt conditions. Reovirus structure is the subject of the last aim. Work will continue from the demonstration that the 3' ends of a least 7 of the 10 RNA segments of reovirus RNA are in intimate contact with viral protein. The proteins involved in this binding or contact will be identified. Structural details of the transcription event that takes place with the virus will be examined, and very old observations that the reovirus RNA genome need not be segmented under certain conditions will be reexamined. The use of a cap-binding protein that has been recently isolated will be explored.
