Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII

Abstract

Some DNA species are resistant towards the restriction endonuclease EcoRII despite the presence of unmodified recognition sites. We show that 14 base-pair oligonucleotide duplexes containing the EcoRII recognition site 5'-CC(A/T)GG are cleaved by this enzyme and are able to stimulate EcoRII cleavage of such resistant DNA molecules (e.g. DNA of bacterial virus T3). A direct correlation between the concentration of oligonucleotide duplex molecules and the degree of EcoRII digestion of the primarily resistant DNA is observed. This indicates a stoichiometric rather than a catalytic mode of enzyme activation. An excess of DNA devoid of EcoRII sites ('non-site' DNA, e.g. MvaI-digested T7 DNA) does not interfere with the activity of EcoRII.

Specificity Changes in the Evolution of Type II Restriction Endonucleases

“…This was confirmed by DNA cleavage experiments in the presence of increasing amounts of an oligodeoxyribonucleotide substrate (Fig. 4) that clearly demonstrate there is no activation in trans, as would have been expected for a Type IIE enzyme (50,51). Furthermore, MboI does not cleave a substrate with two sites more readily than a substrate with one site (data not shown).…”

A Model of EcoRII Restriction Endonuclease Action: The Active Complex is Most Likely Formed by One Protein Subunit and One DNA Recognition Site

“…A feature of endonuclease EcoRII that distinguishes it from many other type II restriction endonucleases is its inability to cleave DNA molecules when substrate recognition sites are very far apart (3). The resistance of such recognition sites to cleavage can be overcome by adding to the reaction mixture short duplexes containing recognition sites or by adding DNA molecules with a high frequency of EcoRII sites (3,5). This resistance can be also overcome by the presence of modi® ed EcoRII sites, which are usually resistant or almost resistant to EcoRII endonuclease cleavage.…”

Crystallization and preliminary X-ray diffraction analysis of restriction endonuclease EcoRII

“…EcoRII is a member of the expanding group of type II restriction endonucleases, subclass IIe, which share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA (Pein et al, 1989;Oller et al, 1991;Petrauskene et al, 1992;Karpova et al, 1993). EcoRII recognizes the nucleotide sequence 5 H -CCWGG (W = A or T) in double-stranded DNA and cleaves it at the 5 H end of the ®rst cytosine base.…”

“…The enzyme is catalytically inactive on single recognition sites in larger DNA molecules and even on two sites in the same DNA (located`cis') when their siteseparation distance exceeds a critical length. Hydrolysis of these a priori resistant DNA substrates can be achieved by the addition of (i) susceptible EcoRII sites (Kru È ger et al, 1988) or (ii) a short synthetic EcoRII recognition site containing oligonucleotide duplexes (Pein et al, 1989;Petrauskene et al, 1992). The activation of enzyme occurs when the EcoRII endonuclease dimer interacts with two DNA recognition sites (Karpova et al, 1993;Petrauskene et al, 1994).…”