Type II restriction endonucleases: structure and mechanism

Abstract

Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few restriction endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II restriction endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.

Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

“…The properties of a recently characterized restriction enzyme, BbvCI (30,31), yielded a general method for analyzing the diffusional translocation of proteins on DNA. Many restriction enzymes are dimers of identical subunits that recognize palindromic sequences (4,5), but BbvCI is a heterodimer that recognizes a nonpalindromic site. The restriction enzymes that recognize nonpalindromic targets are often monomers (5), and two monomers, each bound to a separate recognition site (38), have to associate to cut the DNA.…”

“…For example, it could be applied to a repair enzyme that removes damaged bases from DNA, by examining its processivity on substrates with two target bases, either one in each strand or two in one strand. Likewise, it could be used on enzymes that act at hemi-methylated sites, by constructing DNA substrates where either some sites are methylated in one strand and others in the opposite strand or where one strand is methylated at all sites and the other fully unmethylated, as is the case in vivo after the semiconservative replication of fully methylated DNA (4,5).…”

“…DNA-protein interaction ͉ recognition sequence ͉ restriction enzyme G enetic events such as replication, transcription, and restriction depend on proteins acting at specific DNA sites (1)(2)(3)(4)(5). The specific sequences constitute a minute fraction of genomic DNA so the protein is unlikely to collide with its target site by random diffusion through bulk solution (6,7).…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…They occur in a wide variety of unicellular organisms, including eubacteria and archaea (1,2), and comprise two contrasting enzymatic activities: a restriction endonuclease (REase) and a methyltransferase (MTase). The REase recognizes and cleaves foreign DNA sequences at specific sites, while MTase activity ensures discrimination between self and nonself DNA, by transferring methyl groups to the same specific DNA sequence within the host's genome (Fig.…”

“…Generally, type II REases are homodimeric or homotetrameric and cleave DNA within or near their target site. These enzymes are highly diverse and are known to utilize at least five types of folds: PD-(D/E)XK, PLD, HNH, GIY-YIG, and halfpipe, e.g., R.EcoRI, R.BfiI, R.KpnI, R.Eco29kI, and R.PabI enzymes, respectively (2,(7)(8)(9)(10). Type II enzymes are the most widely studied and are also extensively utilized nucleases in genetic engineering.…”