Role of superhelicity in homologous pairing of DNA molecules promoted by Escherichia coli recA protein.
In the presence of ATP and an excess of recA protein, superhelical closed circular DNA (form I DNA) and homologous single-stranded fragments paired to form D-loops in the early stage of incubation and dissociated during subsequent incubation. RecA protein that was not bound to single-stranded DNA ("free recA protein") was shown to be responsible for the dissociation of D-loops. Larger amount of free recA protein gave a lower final yield of D-loops. When the concentration of form I DNA was increased in the presence of a fixed amount of single-stranded DNA, larger amounts of free recA protein were required to produce a certain extent of dissociation. When form I DNA, excess recA protein, and ATP were incubated without single-stranded DNA, or with heterologous single-stranded fragments before the addition of homologous single-stranded fragments, formation and subsequent dissociation of D-loops were observed as in the case when all components of the reaction were added from the start. Therefore, the dissociation of D-loops is a result of the stoichiometric interaction between free recA protein and form I DNA bearing D-loops. In the process of formation and dissociation of D-loops, form I DNA was converted to an inactive substrate without any apparent damage to the DNA. The concentration of free recA protein appeared to decrease during the reaction. These observations revealed that formation and dissociation of D-loops are sequential reactions when form I DNA is the substrate and recA protein is present in excess. The dissociation of D-loops and the inactivation of form I DNA can be explained by a model in which recA protein cooperatively binds to form I DNA from the site of D-loop, resulting in stimulation of unidirectional unwinding of the double helix.
D001426:Bacterial Proteins / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011938:Rec A Recombinases; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D011938:Rec A Recombinases