Activation of restriction endonucleaseEcoRII does not depend on the cleavage of stimulator DNA

Abstract

The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.

Kinetic Analysis of the Coordinated Interaction of SgrAI Restriction Endonuclease with Different DNA Targets

“…It has been shown that all endonucleases requiring two sites for catalysis cleave plasmid substrates with a single site less efficiently than those with two sites. The cleavage efficiency of solitary sites, however, may be increased by providing a second site on a short oligonucleotide duplex in trans (22,27,42). SgrAI did not follow this pattern, because the canonical sites provided on oligonucleotide duplexes inhibited the cleavage of a plasmid substrate containing a single canonical site (34).…”

“…In an EcoRII complex that is bound to two copies of its recognition sequence, only one sequence is cleaved, whereas the other sequence serves as an allosteric activator (23, 24, 26 -28). The products of the EcoRII reaction (cleaved sites) may also serve as an allosteric activator by stimulating the cleavage activity of EcoRII at the refractory sites (27). In this respect, SgrAI resembles EcoRII, because it also can use the reaction products to assist in cleavage at other sites; however, there is a major difference between the two enzymes.…”

“…For example, Type IIE endonucleases such as NaeI and EcoRII are homodimeric proteins that have two distinct DNA binding clefts (18 -20) and thus are capable of binding two copies of their recognition sequence per protein dimer. However, only one of these recognition sequences is cleaved while the other serves as an allosteric effector (21)(22)(23)(24)(25)(26)(27)(28). Studies with NaeI reveal that the recognition sequences flanked by G/C-rich regions are preferred by the activator site, whereas the catalytic site prefers A/T-rich regions (22,25).…”

“…Studies with NaeI reveal that the recognition sequences flanked by G/C-rich regions are preferred by the activator site, whereas the catalytic site prefers A/T-rich regions (22,25). In the case of EcoRII, it has been shown that some non-cleavable or pre-digested recognition sequences can be bound at the activator site and stimulate the cleavage of refractory sites (27).…”

A new crystal form of restriction endonuclease EcoRII that diffracts to 2.8 Å resolution

“…The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. The activation of enzyme occurs when an EcoRII dimer interacts with two DNA-recognition sites (Kru È ger et al, 1988;Pein et al, 1991;Gabbara & Bhagwat, 1992;Petrauskene et al, 1994).…”