RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.
The importance of 3' single-stranded ends in homologous recombination led us to reevaluate reactions of single strands at homologous sites in the interior of linear duplex DNA. As the length of homology increased, the yield of joints increased up to about 2 kb of homology, at which the apparent yields were the same at either 3' or 5' single-stranded ends, or away from ends, although such joints were qualitatively different. In the presence of RecA protein and ATP, joints that formed between any of these single strands and interior sequences in duplex DNA were in a dynamic state in which they constantly recycled. Consequently, their apparent yields at steady state were sensitive to conditions of reaction, such as the concentrations of DNA, salt, and ATP vs ADP, observations which rationalize conflicting reports in the literature. The dynamic state of joints in the interior of duplex DNA strengthens the conclusion [see Burnett et al. (1994)] that RecA protein dissociates joints when it cannot displace the 5' end of a strand from the recipient duplex.
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