Salt-Stable Complexes of theEscherichia coliRecBCD Enzyme Bound to Double-Stranded DNA

We have examined binding of the RecBCD enzyme to linear double-stranded DNA under two types of conditions. Binding in the absence of ATP can be measured by a nitrocellulose filter binding assay or by gel retardation on polyacrylamide gels. The binding is tightest to ends with four-nucleotide single-stranded 3′-termini (3′-overhang > 5′-overhang > blunt ends). The Kdfor blunt ends is 3.0 (±0.5) nM, in 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2. The binding is weakened in the presence of NaCl, with none detected at 0.5 M NaCl. Binding in the presence of ATP and low MgCl2concentrations (“unwinding conditions”) can be measured by the filter assay and on agarose gels. Enzyme–DNA complexes allowed to form under unwinding conditions are not affected by 0.5 M NaCl. ATP hydrolysis continues and the complexes dissociate at a rate similar to those to which no salt is added. These enzyme–DNA complexes can be trapped with EDTA, and they are unaffected for at least 1 h by 0.5 M NaCl or heparin. The results show that the enzyme–DNA interactions are different when the enzyme is bound to partially unwound DNA compared to when it is bound to the ends of fully duplex DNA.