Abstract
Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formation and dissociation of D-loops from negative superhelical closed circular double-stranded DNA (form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the form I DNA without apparent damage to the DNA. The dissociation of D-loops is accompanied by hydrolysis of ATP to ADP that apparently depends on homologous DNA molecules (homology-dependent ATP hydrolysis). However, at a lower concentrations of ATP, we observed anomalous kinetics in the formation and dissociation of D-loops; as the concentration of ATP was decreased, there was a progressively smaller dissociation of D-loops and a faster resynthesis in the second phase, without changing the rate of the first formation of D-loops. This anomaly might suggest that, as the increase in the amount of ADP relative to that of ATP, dissociation form I DNA is stimulated before formation of D-loops is inhibited. We found that addition of ADP inhibited competitively both formation and dissociation of D-loops and that the latter process was more sensitive to the inhibition than was the former process. Addition of a sufficient amount of ADP to inhibit both formation and dissociation of D-loops, cessation of homology-dependent hydrolysis of ATP, or incubation at low temperature resulted in reactivation of form I DNA that had been inactivated by the sequence. In the presence of an ATP-regenerating system, we confirmed our previous result that limiting the amount of recA protein also causes anomalous kinetics in the formation and dissociation of D-loops. These observations indicate that the formation and dissociation of D-loops and the inactivation and reactivation of form I DNA make a circular reaction sequence.
Highlights
Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formationand dissociation of D-loopsfrom negative superhelical closed circular double-stranded DNA(form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the formI DNA without apparent damage to the DNA
Addition of a sufficient amount o f A D Pto inhibit both formation and dissociation of Dloops, cessation of homology-dependent hydrolysis of ATP, or incubation at low temperature resulted in reactivation of formI DNA that had been inactivated by the sequence
Anomalous Kinetics in the Formation of D-loops by Limiting the Amount of ATP-When form I DNA and homologous single-stranded fragments are incubated with excessrecA protein in the presence of ATP, D-loops are formed initially and dissociate completely; in the presence of 0.40 ~ L M single-stranded fragments and 1.3 mM ATP, formation of Dloops reaches its peak at about 4 min, and subsequent dissociation is complete by 30 min (Figs. 2A and 4B; see Ref. 37)
Summary
Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formationand dissociation of D-loopsfrom negative superhelical closed circular double-stranded DNA(form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the formI DNA without apparent damage to the DNA. In the presence of an ATP-regenerating system, we confirmed our previous result that limiting the amount of recAprotein causes anomalous kinetics in the formation and dissociation ofD-loops These observations indicate that the formation and dissociation of D-loopsand the inactivation and reactivation of formIDNAmak ea circular reaction sequence. We found that, with formI DNA’ and homologous single-stranded fragments as substrates, recA protein, when present in excess, formed D-loops, subsequently dissociated them, and converted thfeorm I DNA to an inactive substrate for reinitiation of D-loop formation without apparent damage to the DNA (Ref. 37; Fig. 1, steps Z and ZI). The homology-dependent ATP hydrolysis is related to thedissociation of D-loops and theinactive state of form I DNA rather than the formatioonf D-loops [38].In this paper, we will present evidence for a pathway by which the form I DNA inactivated through formation and dissociation of D-loops is reactivated and can be used as a substrate for a new sequence of formation and dissociation (Fig. 1, step ZZI)
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