RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process.
Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease. The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length.
Rao
B J
BJ
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510.
Jwang
B
B
Radding
C M
CM
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013696:Temperature
D004269:DNA, Bacterial; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D000251:Adenosine Triphosphatases