Effect of Deoxyribonucleic Acid Length on the Adenosine Triphosphatase Activity of Escherichia coli Restriction Endonuclease B

Abstract Restriction endonuclease B possesses a DNA-dependent ATPase activity. The ATP hydrolysis can continue for hours after the DNA hydrolysis has stopped and is due to a stable DNA-enzyme complex. RFI of bacteriophage f1 is much more active than its full length linear form (RFIII) in stimulating the ATP hydrolysis. Experiments with λ phage DNA sheared into various size molecules indicate that the ATPase activity is a direct function of the molecular weight of linear DNA in a range between 0.9 to 13 x 106 daltons. While sonicated, unmodified λ DNA molecules (5 x 105 daltons) fail to stimulate the ATPase activity, they do inhibit the intact DNA-stimulated ATP hydrolysis. The results can be interpreted as follows. (a) Endonuclease R-B recognizes DNA at the SB sites; this recognition step is independent of DNA length. (b) The probability that a linear DNA molecule is cleaved by an enzyme molecule bound to the DNA depends upon the length of the DNA: the greater the number of nucleotide pairs the higher the probability of cleavage. Circularization of a small DNA duplex also increases the probability of cleavage. One possible explanation is that the enzyme travels along the DNA molecule before cleaving the DNA. If the enzyme reaches an end of the DNA molecule before cleavage occurs, the enzyme molecule is inactivated. (c) After DNA hydrolysis has occurred, the enzyme (or one of its components) remains on the DNA molecule and causes the massive ATP hydrolysis.