Abstract
The various activities of the recBC enzyme of Escherichia coli are differentially sensitive to reaction conditions. Although spermidine up to 50 mM and putrescine up to 40 mM in the presence of Mg/sup 2 +/ produce no significant alteration in the activities of the enzyme, low concentrations of Ca/sup 2 +/ have a dramatic effect. In the presence of 1 mM Ca/sup 2 +/, 1 mM Mg/sup 2 +/, and 5 mM ATP, the DNA-dependent ATPase is unaffected, but the double strand DNA exonuclease and the single strand DNA exonuclease and endonuclease activities are completely inhibited. Duplex phage T7 DNA which has been exposed to enzyme under these conditions remains wholly duplex and full length, although about 5 nicks are made randomly in each duplex. When DNA binding protein from E. coli is included in the reaction with enzyme and DNA, the products are, at early stages of the reaction, duplex DNA molecules with one or two single-stranded tails at a terminus; after prolonged incubation, solely single-stranded fragments of 5000 to 35,000 nucleotides are found. (Full length strands are roughly 40,000 nucleotides long.) These products can be rationalized by a model in which the enzyme binds initially to the terminusmore » of a duplex DNA molecule and then tracks along the DNA, unwinding the strands of the helix as it moves. This denaturation would be transient and localized around the enzyme molecule so that the DNA would renature after the enzyme has passed through a particular region. However, the renaturation can be prevented by the presence of DNA binding protein during the reaction. During the passage of an enzyme molecule through a DNA molecule, a limited number of nicks are randomly introduced in a manner essentially unaffected by binding protein. This mode of action is distinct from that observed for the degradation of duplex DNA with Mg/sup 2 +/ but not Ca/sup 2 +/ present.« less
Highlights
The various activities of the recBC enzyme of Escherichia coli are differentially sensitive to reaction conditions
When DNA binding protein (DBP) was added to the reactions containing Ca”+ and Mg’+, the DNA sedimented in neutral sucrose in a fashion comparable to the sedimentation of denatured T7 DNA under these conditions (Fig. 2, A and C), which suggests that the recBC enzyme unwinds the duplex
Ca’ ’ and Mg’+ present, the DNA was found as single-stranded fragments of 5,000 to 35,000 nucleotides:’ it would seem that the recBC enzyme makes a limited number of nicks in each strand of the T7 DNA molecule as it unwinds the duplex
Summary
Materials-Commercial bacterial alkaline phosphatase (Worthington) was freed of nuclease on DEAE-cellulose [16]. Glycerol gradient fraction of recBC enzyme was used throughout; its purifica~on and assav for DNase and ATPase activities have been described. Tris-HCl (pH 8.1), 5 mM EDTA, and 2.5 M NaCl. Hieh salt concentrations were used to remove protein from the DNA. After incubating at 37°C for 30 min, excess bacterial alkaline phosphatase was added and the reaction mixtures incubated for an additional 30 min at 37’C. This treatment hydrolyzes all remaining ATP, thereby preventing any recBC enzyme activity in subsequent incubations. The singlestranded DNA formed in the first incubation with recBC enzyme was digested by incubating at 37°C for 30 min with excess E. coli exonuclease I in 10 mM MgC12. The acid-soluble nucleotides formed in this way were measured as described previously [3]
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