Figure 3.

ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α-³³P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by HindIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by PstI; ▴, Δ (triangles), linear dsDNA with blunt ends generated by HincII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl₂ (the standard reaction mixtures for homologous joint formation by RecA: (31)); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl₂, without PEG).