DNase protection by recA protein during strand exchange. Asymmetric protection of the Holliday structure.
When recA protein pairs linear duplex DNA with a homologous duplex molecule that has a single-stranded tail, it produces a recombination intermediate called the Holliday structure and causes reciprocal or symmetric strand exchange, whereas the pairing of a linear duplex molecule with fully single-stranded DNA leads to an asymmetric exchange. To study the location of recA protein on DNA molecules undergoing symmetric exchange, we labeled individually each end of the four strands involved and looked for protection against DNase I or restriction endonucleases. As expected, because of its preferred binding to single-stranded DNA, recA protein protected the single-stranded tails of either substrates, or products. In addition however, strong protection extended into the newly formed heteroduplex DNA along the strand to which recA protein was initially bound. Experiments with uniformly labeled DNA showed a corresponding homology-dependent asymmetry in the protection of the tailed substrate versus its fully duplex partner. Restriction experiments showed that protection extended 50-75 base pairs beyond the point where strand exchange was blocked by a long region of heterology. When compared with earlier observations (Chow, S. A., Honigberg, S. M., Bainton, R. J., and Radding, C. M. (1986) J. Biol. Chem. 261, 6961-6971), the present experiments reveal a pattern of association of recA protein with DNA that suggests a common mechanism of asymmetric and symmetric strand exchange.
Chow
S A
SA
Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Honigberg
S M
SM
Radding
C M
CM
D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D011938:Rec A Recombinases; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I