The transfer of strands from one DNA molecule to another is common to all recombination events; in the homologous pathway of E. coli, two proteins, RecA and SSB orchestrate this transfer of strands. First, joint structures are formed between two duplex DNAs (and must contain a gap), and next, a single strand is transferred from one DNA to the other; this requires a free end. RecA forms the basic scaffold to which the DNAs are held and unwound. The role of SSB, a single strand DNA binding protein, is less well understood. Also we know little about the arrangement of the DNA in th nucleoprotein complexes undergoing recombination. To understand the mechanism of recombination, we must know how the DNAs are held and become unwound on these protein scaffolds. In pursuit of this goal, our group has been investigating how SSB and RecA alone arrange single stranded and duplex DNA. We demonstrated that SSB arranges single stranded DNA into beaded nucleosomal chains and showed that RecA forms several different but probably related nucleoprotein fibers with both single stranded and duplex DNA. We will continue these studies, so that we can relate the ultrastructure of each complex to the others. We will determine exactly how the RecA monomers are arranged in each fiber, and measure the unwinding of DNA by RecA when the ATP analog, ATP and S, is present. This will test the idea that there is one RecA filament common to all of these structures and that it extends 1.5 fold during strand transfer. We will examine the competition of RecA, SSB and mutants thereof for a single stranded DNA using an EM and antibody staining technique. This will provide structural insights into how SSB and RecA cooperate in recombination. Next, the ultrastructure of nucleoprotein complexes containing intermediates in joint formation and strand transfer reactions in vitro will be characterized using our direct EM methods including antibody stains. We will examine the location of SSB in the complexes, determine which of the RecA filament structures is present, and how many DNA strands are associated with each RecA filament. Ultimately these studies must be correlated with direct visualization of analagous complexes engaged in recombination in the living cell. Using our gentle lysis technique for disrupting cells on the EM grid we will begin this work.
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