Abstract
The complete exchange of strands between circular single-stranded and full length linear duplex DNAs promoted by the recA protein of Escherichia coli is dependent upon the hydrolysis of ATP and is strongly stimulated by the single-stranded DNA binding protein (SSB). In the presence of SSB, stable complexes of recA protein and single-stranded DNA are formed as an early step in the reaction. These complexes dissociate when the ADP/ATP ratio approaches a value of 0.6-1.5, depending upon reaction conditions. Thus, ATP hydrolysis never proceeds to completion but stops when 40-60% of the input ATP has undergone hydrolysis. recA protein can participate in a second round of strand exchange upon regeneration of the ATP. While 100-200 mol of ATP are hydrolyzed/mol of heteroduplex base pair formed under standard reaction conditions in the presence of SSB, this value is reduced to 16 at levels of ADP lower than that required to dissociate the complexes. ATP hydrolysis appears to be completely irreversible since efforts to detect exchange reactions using 18O probes have been unsuccessful.
Highlights
The complete exchange of strands between circular single-stranded and full length linear duplexDNAs promoted by the re;cA protein (recA) protein of Escherichia coliis dependent upon the hydrolysis of ATP andis strongly stimulatedby the single-stranded DNA bindingprotein (SSB)
The recA protein of Escherichia coli promotes DNA strand exchange reactions that can take a variety of forms [1,2,3,4].A informative reaction is the exchange of strands between circular + X SSDNA'and full length linear $X duplex DNA, which produces a replicative form 11-like product and a displaced linear (+) single strand [3, 5,6].The reaction can be divided kinetically into two phases, an initial pairing to form a short region of heteroduplex followed by branch migration to extend the heteroduplex
Perhaps the most unusual characteristic of recA protein-promoted ATP hydrolysis is its limited extent in the presence of duplex DNAat pH 6.2 [13].Under these conditions, recA protein catalyzes the hydrolysis of only 60%of the ATP provided in the reaction, an effect that is largely independent of ATP concentration
Summary
The assay for heteroduplex formation, based on the conversion of 3H-labeledcircular +X ssDNA into an S1nuclease-resistant form, has been described [6]. Where measurements of extent are reported, the values represent the average of a t least three determinations takenbetween 40 and 60 min after the startof the reaction. Where both labeled and unlabeled +X ssDNA were present, “per cent heteroduplex” refers to the fraction of ‘H-labeled DNA incorporated into heteroduplex rather than the fraction of the total DNA that had reacted. Data were treated by adding counts/min in the ADP andATP, multiplying the total by the background percentage to determine the total background counts/min in that sample. Aliquots were taken from the fractions and counted in a liquid scintillation counter
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