Production of triple-stranded recombination intermediates by RecA protein, in vitro.
During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.
Rao
B J
BJ
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510.
Jwang
B
B
Dutreix
M
M
D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005090:Exodeoxyribonucleases; D009692:Nucleic Acid Heteroduplexes / Q000096:biosynthesis; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I