PMID:9473301
| Citation |
Gabbidon, MR, Rampersaud, VE and Julin, DA (1998) Salt-stable complexes of the Escherichia coli RecBCD enzyme bound to double-stranded DNA. Arch. Biochem. Biophys. 350:266-72 |
|---|---|
| Abstract |
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 Kd for 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 MgCl2 concentrations ("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. |
| Links |
PubMed Online version:10.1006/abbi.1997.0530 |
| Keywords |
Adenosine Triphosphate/metabolism; Adenosine Triphosphate/pharmacology; DNA/metabolism; DNA Helicases/metabolism; DNA-Binding Proteins/metabolism; Edetic Acid/pharmacology; Escherichia coli/enzymology; Exodeoxyribonuclease V; Exodeoxyribonucleases/metabolism; Heparin/pharmacology; Single-Strand Specific DNA and RNA Endonucleases/metabolism; Sodium Chloride/pharmacology |
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