Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+.

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