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Type II Restriction Endonucleases : a Historical Perspective and More Published online 30 May 2014 Nucleic Acids Research, 2014, Vol. 42, No. 12 7489–7527 doi: 10.1093/nar/gku447 SURVEY AND SUMMARY Type II restriction endonucleases––a historical perspective and more Alfred Pingoud1,*,†, Geoffrey G. Wilson2,† and Wolfgang Wende1 1Institute of Biochemistry, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany and 2New England Biolabs Inc., 240 County Road, Ipswich, MA 01938-2723, USA Downloaded from Received January 7, 2014; Revised May 02, 2014; Accepted May 7, 2014 ABSTRACT combat viral infections, these enzymes allow unmanageable http://nar.oxfordjournals.org/ tangles of macromolecular DNA to be transformed with This article continues the series of Surveys and Sum- unsurpassable accuracy into convenient, gene-sized pieces, maries on restriction endonucleases (REases) begun a necessary first step for characterizing genomes, sequenc- this year in Nucleic Acids Research.Herewedis- ing genes, and assembling DNA into novel genetic arrange- cuss ‘Type II’ REases, the kind used for DNA anal- ments. It seems unlikely that today’s Biomedical Sciences ysis and cloning. We focus on their biochemistry: and the Biotechnology industry would have developed with- what they are, what they do, and how they do it. Type out Type II restriction enzymes, and certainly not at the II REases are produced by prokaryotes to combat startling pace we have witnessed since their discovery only at Bibliothekssystem der Universitaet Giessen on February 10, 2015 bacteriophages. With extreme accuracy, each recog- a few decades ago. nizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The INTRODUCTION discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since im- Several reviews of restriction endonucleases (REases) have pacted every corner of the life sciences. They be- appeared as Surveys and Summaries in Nucleic Acids Re- came the enabling tools of molecular biology, ge- search recently. These concerned the somewhat esoteric Type I (1), Type III (2) and Type IV (3) REases; highlights netics and biotechnology, and made analysis at the of half a century of REase research and discovery (4); and most fundamental levels routine. Hundreds of dif- the connection between REases and genetic addiction sys- ferent REases have been discovered and are avail- tems (5). The present review focuses on the more familiar, able commercially. Their genes have been cloned, Type II REases, the ‘work horses’ (6) of modern molec- sequenced and overexpressed. Most have been char- ular biology, used daily in laboratories for DNA analysis acterized to some extent, but few have been studied and gene cloning. This review is partly historical, as were in depth. Here, we describe the original discoveries the others, and emphasizes the importance of the enzymes in this field, and the properties of the first Type II EcoRI and EcoRV, among the first REases discovered, REases investigated. We discuss the mechanisms and the two most thoroughly studied (Figure 1). It is also of sequence recognition and catalysis, and the var- partly contemporary, and provides an up-to-date overview ied oligomeric modes in which Type II REases act. of the field, although one that is necessarily not compre- hensive. Over 350 different Type II prototype REases are We describe the surprising heterogeneity revealed known, each unique in its biochemistry, and with its own by comparisons of their sequences and structures. story to tell. For most of these, anywhere from a few to over one hundred similar enzymes from sequenced organ- isms are known, some characterized but most putative. And PROLOGUE REBASE (rebase.neb.com/rebase/rebase.html), the defini- We wonder what Molecular Biology would look like today tive source for information on REases and their compan- had Type II restriction enzymes not been discovered. Syn- ion proteins (7), lists over 8000 research publications in this thesized in bewildering variety by bacteria and archaea to field, too many by far to be discussed here. We apologize in *To whom correspondence should be addressed. Tel: +49 641 35401; Fax: +49 641 35409; Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 7490 Nucleic Acids Research, 2014, Vol. 42, No. 12 Number of EcoRI Publications Number of EcoRV Publications 60 50 40 30 Downloaded from 20 http://nar.oxfordjournals.org/ 10 0 at Bibliothekssystem der Universitaet Giessen on February 10, 2015 Figure 1. Number of publications for EcoRI and EcoRV per year from 1972 to 2012. Only publications are listed in which EcoRI and EcoRV are listed in the title. Source: REBASE (7). advance for our omissions. For a broader review of Type II R.AciI), an REase; M1∼M2.AciI (or M.AciI), a compos- REases see Pingoud et al. (8). A comprehensive collection of ite, double MTase, and C.AciI, a control protein. REases reviews on REases has been published as a book: Pingoud that recognize the same DNA sequence, regardless of where (Ed.) REases (9). Two excellent additional reviews describe they cut, are termed ‘isoschizomers’ (iso = equal; skhizo early work on Type II REases by Modrich & Roberts (10) = split) (13). Isoschizomers that cut the same sequence at and Roberts & Halford (11). different positions are further termed ‘neoschizomers’ (neo Following the original proposal by Smith and Nathans = new) (14). Isoschizomers that cut at the same position (12), restriction enzymes are named according to the tax- are frequently, but not always, evolutionarily drifted ver- onomy of the organism in which they were discovered. The sions of the same enzyme (e.g. BamHI and OkrAI). Invari- first letter of the enzyme refers to the genus of the organism ably, neoschizomers are different enzymes altogether (e.g. and the second and third to the species. This is followed by EcoRII and MvaI). letters and/or numbers identifying the isolate. Roman nu- Like the other types of restriction enzymes, Type II merals are used, finally, to specify different enzymes from REases occur exclusively in unicellular microbial life the same organism. For example, the enzyme ‘HindIII’ was forms––mainly bacteria and archaea (prokaryotes)––and discovered in Haemophilus influenzae, serotype d, and is dis- are thought to function primarily to protect these cells tinct from the HindI and HindII endonucleases also present from viruses and other infectious DNA molecules. A in this bacterium. The DNA-methyltransferases (MTases) group of large viruses that infect the eukaryotic algae, that accompany restriction enzymes are named in the same Chlorella, also encode Type II REases (15,16) and DNA- way, and given the prefix ‘M.’. When there is more than methyltransferases (MTases; (17)). The genes for Type II one MTase, they are prefixed ‘M1.’, ‘M2.’, if they are sepa- REases occur mainly on chromosomes, and occasionally on rate proteins and ‘M.’ or ‘M1∼M2.’ when they are joined. transmissible elements such as plasmids, transposons and REases are designated explicitly by the prefix ‘R.’; this is insertion sequences. They rarely occur on bacteriophages, usually omitted when there is no ambiguity. Enzymes in although MTases sometimes do, as one of several forms of which restriction and modification activities occur in the viral self-protection (18–20). In the discussions that follow, same polypeptide chain are prefixed ’RM.’ (e.g. RM.BcgI), we refer to all of these sources loosely, as ‘prokaryotes’, or which again is omitted when there is no ambiguity. Ad- ‘microbes’. Type II REases are more heterogeneous than the ditional proteins are prefixed ‘V.’ (for Vsr endonucleases) other REase types in part because ‘Type II’ is a utilitarian and ‘C.’ (for control proteins). For example, the AciI R- classification, based on enzymatic behavior rather than phy- M system, from Arthrobacter citreus, comprises AciI (or logeny.Type II REases are a conglomeration of many differ- Nucleic Acids Research, 2014, Vol. 42, No. 12 7491 ent proteins that, by definition, have the common ability to cleave duplex DNA at a fixed position within, or close to, their recognition sequence. This cleavage generates repro- ducible DNA fragments, and predictable gel electrophoresis patterns, properties that have made these enzymes invalu- able reagents for laboratory DNA manipulation and investi- gation. Almost all Type II REases require divalent cations– –usually Mg2+––as essential components of their catalytic sites. Many can use Mn2+ in place of Mg2+, and a few can use a variety of cations including Co2+,Zn2+,Ni2+ and Cu2+ instead (21). Ca2+ ions usually, but not always, inhibit catal- ysis. A few REases require Zn2+ ions (e.g. BslI, PacI and 2+ DpnI (22–24)), or less often Fe ions (e.g. NotI (25)), for in- Downloaded from corporation into Cys4 structural motifs. And a diverse sub- class that catalyze DNA methylation in addition to cleavage (the Type IIG enzymes, discussed later) require the cofactor Figure 2. Hamilton Smith and Daniel Nathans at the Nobel Prize press S-adenosylmethionine (AdoMet or SAM), often for both conference, 12 October 1978 (reproduced with permission from Susie activities. Much of what we know about Type II enzymes Fitzhugh). Original Repository: Alan Mason Chesney Medical Archives, was discovered first with EcoRI and EcoRV. These REases Daniel Nathans Collection. http://nar.oxfordjournals.org/ are representative of the Type IIP subclass that recognize palindromic (symmetric) DNA sequences and generally act as homodimers or homotetramers. Type IIP REases are the K or B.
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