Separation of the presynaptic and synaptic phases of homologous pairing promoted by recA protein.

Homologous pairing of single strands with duplex DNA promoted by recA protein occurred without a lag only when the protein was preincubated with ATP and single-stranded DNA. The rate-limiting presynaptic interaction of recA protein and single strands showed a high temperature coefficient: it proceeded 30 times more slowly at 30 degrees C than at 37 degrees C, whereas synapsis showed a normal temperature coefficient. Thus, the presynaptic phase could be separated experimentally from the rest of the reaction by preincubation of single strands with recA protein and ATP at 37 degrees C, followed by a shift to 30 degrees C before double-stranded DNA was added. The presynaptic phase was an order of magnitude more sensitive to inhibition by ADP than was subsequent strand exchange. Presynaptic complexes that were formed at 37 degrees C decayed only slowly at 30 degrees C, but Escherichia coli single strand binding protein caused complexes to form rapidly at 30 degrees C which indicates that single strand binding protein accelerated the rate of formation of complexes. Preincubation synchronized the initial pairing reaction, and further revealed the rapid formation of nascent heteroduplex DNA 250-300 base pairs in length.