EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0.5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.Concerning their substrate requirements, certain restriction endonucleases (ENases) 1 that interact at a distance with at least two DNA recognition sites resemble proteins involved in processes like transcription control, DNA recombination, and replication in both prokaryotes and eukaryotes (1-6). In addition to EcoRII, the first ENase for which this special characteristic was described (Ref. 7; for a review see 8), comprehensive studies were also performed on NaeI. Electron microscopy of DNA-NaeI complexes revealed loop structures connecting NaeI sites on the same DNA molecule (9). Another extraordinary type II ENase is SfiI, whose tetrameric enzyme structure bridges two specific recognition sites and cleaves them simultaneously (10, 11). A few other ENases are considered to belong to this particular ENase group (12, 13) termed IIe, characterized by requiring a second DNA (effector) site to perform cleavage (14).The complex mode of action of these particular ENases appears to limit their efficiency in DNA restriction and may reflect additional biological functions besides the destruction of invading DNA. Other type II ENases (non IIe) like EcoRI and EcoRV use the simpler and more powerful mechanism of linear diffusion along the helical pitch of the DNA that enables them to find every single recognition site and effectively restrict foreign DNA (15, 16). Some experimental results support the idea of an evolutionary relationship of type IIe ENases with other protein families; Jo and Topal (17) discovered a sequence motif shared by ENase NaeI and the active site of human DNA ligase where the essential lysine 43 of the ligase is replaced by leucine in NaeI. A single amino acid substitution, L43K, in NaeI converts the ENase to a DNA topoisomerase with pref...

Target sequence-specific DNA binding regions of the restriction endonuclease EcoRII were identified by screening a membrane-bound EcoRII-derived peptide scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex. Dodecapeptides overlapping by nine amino acids and representing the complete protein were prepared by spot synthesis. Two separate DNA binding regions, amino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by exchanging every residue of both binding sites for all other amino acids demonstrated that replacing basic residues in the consensus motifs significantly reduced DNA binding. EcoRII mutant enzymes generated by substituting alanine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structure was unaffected. Furthermore, Glu96, which is part of a potential catalytic motif and also locates to DNA binding site I, was demonstrated to be critical for DNA cleavage and binding. Homology studies of DNA binding site II revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionally related DNA binding sites in diverse restriction endonucleases with recognition sequences containing terminal C:G or G:C pairs.

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