NAR Breakthrough Article

SURVEY AND SUMMARY

Highlights of the DNA cutters a short history of therestriction enzymesWil A M Loenen1 David T F Dryden2 Elisabeth A Raleigh3 Geoffrey G Wilson3

and Noreen E Murrayy

1Leiden University Medical Center Leiden the Netherlands 2EaStChemSchool of Chemistry University ofEdinburgh West Mains Road Edinburgh EH9 3JJ Scotland UK and 3New England Biolabs Inc 240 CountyRoad Ipswich MA 01938 USA

Received August 14 2013 Revised September 24 2013 Accepted October 2 2013

ABSTRACT

In the early 1950rsquos lsquohost-controlled variation inbacterial virusesrsquo was reported as a non-hereditaryphenomenon one cycle of viral growth on certainbacterial hosts affected the ability of progeny virusto grow on other hosts by either restricting orenlarging their host range Unlike mutation thischange was reversible and one cycle of growth inthe previous host returned the virus to its originalform These simple observations heralded the dis-covery of the endonuclease and methyltransferaseactivities of what are now termed Type I II III andIV DNA restriction-modification systems The Type IIrestriction enzymes (eg EcoRI) gave rise to recom-binant DNA technology that has transformed mo-lecular biology and medicine This review traces thediscovery of restriction enzymes and their continuingimpact on molecular biology and medicine

INTRODUCTION

Restriction endonucleases (REases) such as EcoRI arefamiliar to virtually everyone who has worked withDNA Currently gt19000 putative REases are listed onREBASE (httprebasenebcom) (1) REases are classifiedinto four main types Type I II III and IV with subdiv-isions for convenience almost all require a divalent metalcofactor such as Mg2+ for activity (Table 1 and Figure 1)

Type II REases represent the largest group of characterizedenzymes owing to their usefulness as tools for recombinantDNA technology and they have been studied extensivelyOver 300 Type II REases with gt200 different sequence-specificities are commercially available Far fewer Type IIII and IV enzymes have been characterized but putativeexamples are being identified daily through bioinformaticanalysis of sequenced genomes (Table 1)Here we present a non-specialists perspective on import-

ant events in the discovery and understanding of REasesStudies of these enzymes have generated a wealth ofinformation regarding DNAndashprotein interactions andcatalysis protein family relationships control of restric-tion activity and plasticity of protein domains as wellas providing essential tools for molecular biologyresearch Discussion of the equally fascinating DNA-methyltransferase (MTase) enzymes that almost alwaysaccompany REases in vivo is beyond the scope of thisreview but we note that base flipping first discovered inthe HhaI MTase (2) is not confined to these enzymesalone but appears to be a common phenomenon that isalso used by certain REases (3) and in other nucleic acid-binding enzymes (4ndash7)Most research interest has focused on Type I and II

enzymes for historical and practical reasons so thishistory is weighted to their treatment The moleculargenetic and enzymological properties of these have beenextensively reviewed [see eg (8ndash12)] and separate reviewsof the Type I III and IV systems appear elsewhere in thisjournal

To whom correspondence should be addressed Tel +31 85 878 0248 Email wamloenenlumcnlCorrespondence may also be addressed to David T F Dryden Tel +44 131 650 4735 Fax +44 131 650 6453 Email daviddrydenedacukCorrespondence may also be addressed to Elisabeth A Raleigh Tel +1 978 380 7238 Fax +1 978 921 1350 Email raleighnebcomCorrespondence may also be addressed to Geoffrey G Wilson Tel +1 978 380 7370 Fax +1 978 921 1350 Email wilsonNEBcomyNoreen Murray passed away during the early stages of preparing this review which is fondly dedicated to her memory

The authors wish it to be known that in their opinion the first three authors should be regarded as Joint First Authors

Published online 18 October 2013 Nucleic Acids Research 2014 Vol 42 No 1 3ndash19doi101093nargkt990

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercialre-use please contact journalspermissionsoupcom

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THE FIRST HIGHLIGHTS

Discovery of lsquohost-controlled variationrsquo

Many important scientific developments in the first half ofthe 20th century laid the groundwork for to the discoveryof restriction and modification (R-M) These included thediscoveries of radiation and to the ability to incorporateisotopes in living cells the molecular building blocks ofDNA RNA and protein lsquofilterable agentsrsquo (viruses) theisolation of Escherichia coli and other bacteria and oftheir viruses [called (bacterio)phage] and plasmidsEnabling technical advances included development ofelectron microscopy ultracentrifugation chromatog-raphy electrophoresis and radiographic crystallographyKey was the emerging field of microbial genetics whichflourished owing to the discovery of lysogeny conjuga-tion transduction recombination and mutation

Preliminary descriptions of the phenomenon of R-Mwere published by Luria and Human (1952) (13)Anderson and Felix (1952) (14) and Bertani and Weigle(1953) (15) These reports of lsquohost-controlled variation inbacterial virusesrsquo were reviewed by Luria (1953) (16)Host-controlled variation referred to the observationthat the efficiency with which phage infected new bacterialhosts depended on the host on which they previouslygrew Phage that propagated efficiently on one bacterialstrain could lose that ability if grown for even a singlecycle on a different strain The loss was not due tomutation and one cycle of growth on the previous

Table 1 Characterization and organization of the genes and subunits of the four Types of restriction enzymes

Type Type I Type II Type III Type IV

Features Oligomeric REase and MTasecomplex

Require ATP hydrolysis forrestriction

Cleave variably often farfrom recognition site

lsquoDEAD-boxrsquo translocatingREase

bipartite DNA recognitiondomain

Separate REase and MTaseor combinedREaseMTase fusion

Cleave within or at fixedpositions close torecognition site

Many different subtypes

Combined REase+MTasecomplex

ATP required for restrictionCleave at fixed position

outside recognition sitelsquoDEAD-boxrsquo REase

Methylation-dependent REaseCleave at variable distance

from recognition siteCleave m6A m5C hm5C

andor other modifiedDNA

Many different types

Example eg EcoKI eg EcoRI eg EcoP1I No lsquotypicalrsquo example

Genes hsdR hsdM hsdS eg ecorIR ecorIM eg ecoP1IM ecoP1IR eg mcrA mcrBC mrr

Subunits 135 62 and 52 kDa 31 and 38 kDa for EcoRI 106 and 75 kDa forEcoP1I

Unrelated proteins

Proteins REase 2R+ 2M+ S Orthodox REase 2R REase 1 or 2 R+2M VariesMTase 2M+S (plusmn2R) Orthodox MTase M MTase 2M (plusmn2R)

REBASE 104 enzymes 47 genes cloned34 genes sequenced5140 putatives

3938 enzymes 633 genescloned 597 sequenced9632 putatives

21 enzymes 19 genes clonedamp sequenced 1889putatives

18 enzymes amp genes cloned15 sequenced 4822putatives

Type I and II are currently divided in 5 and 11 different subclasses respectively Few enzymes have been well-characterized but based on the currentavalanche of sequence information many putative genes belonging to all Types and subtypes are being identified and listed on the restriction enzymewebsite (httprebasenebcom) The modification-dependent Type IV enzymes are highly diverse and only a few have been characterized in anydetail In each case an example is given of one of the best-characterized enzymes within the different Types I II and III Note that Type II enzymesrange from simple (shown here for EcoRI) to more complex systems (see Table 2 for the diversity of Type II subtypes)REBASE count is as of 16 September 2013 (httprebasenebcomcgi-binstatlist)

Figure 1 Schematic representation of the functional roles filled in dif-ferent ways in R-M systems The functions served include the follow-ing S DNA sequence specificity MT methyltransferase catalyticactivity M SAM binding E endonuclease T translocation Boxesoutline functions that are filled by distinct protein domains Differentcolours indicate different functions while different boxes representdistinct domains Domains in a single polypeptide are abutted andthose in separate polypeptides spaced apart The order of domains ina polypeptide may varymdasheg not all Type IIS enzymes have thecleavage domain at the C-terminus In some cases functions areintegrated with each other eg S and E functions of Type IIP(striped box) in other cases separate domains carry them out egType IIS See Table 2 for the complexity and diversity of Type IIsubtypes The large families of Type I and Type II systems are currentlysubdivided in 5 and 11 different groups respectively The Type Ifamilies are distinguished by homology the Type II groups are distin-guished by catalytic properties rather than sequence homology TypeIV enzymes were initially identified as hydroxymethylation-dependentrestriction enzymes and currently comprise a highly diverse family Twoexamples are shown with and without a translocation domain

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strain returned the virus to its original state once moreR-M systems of all types initially were investigated inthis same way by comparing the lsquoefficiency of platingrsquo(eop number of plaques on the test host divided by thenumber of plaques on a permissive host) on alternatebacterial hosts (17ndash21) Eop values would range from101 to 105 thus indicating that R-M systems wereeffective barriers to the uptake of DNA see (1622ndash26)for early reviews

DNA modification

A decade after these initial reports Werner Arber andDaisy Dussoix using phage lambda as experimentalsystem showed that it was the phage DNA that carriedthe host-range imprint (17) Different specificities could beimprinted concomitantly both by the bacterium itself (bywhat were later recognized to be the Type I EcoKI andEcoBI systems) and by phage P1 in its latent prophagestate (the Type III EcoP1I system) Gunther Stent sug-gested that DNA methylation might be the basis for themodification imprint thus prompting Arber to show thatmethionine was required in the growth medium toproduce the imprint on the DNA (27) This importantfinding coincided with the discovery of RNA-methyltransferase and MTase activities in bacteria thatcatalysed the formation of m5C and m6A (28) Arberrsquosinterest in the biochemical mechanisms of R-M wasdriven in part by insight that R-M enzymes would proveuseful for analysing DNA molecules and DNAndashproteininteractions He concluded a landmark 1965 review ofhost-controlled modification with the following wordslsquoLooking toward future developments it is to behoped that the enzymes involved in production andcontrol of host specificity will be isolated andcharacterized Such studies paralleled with investigationsof the genes controlling R-M and of their expressionshould eventually permit an explanation of the highdegree of strain specificity for example lsquolsquoby a mechanismof recognition of certain base sequencesrsquorsquo If this last ideashould be correct one may further speculate that a restric-tion enzyme might lsquolsquoprovide a tool for the sequence-specific cleavage of DNArsquorsquo rsquo (22) (our double quotes)

Sequence-specific DNA-cleavage

As chance would have it the R-M systems studied byArber Type I and Type III (25) do not provide simpleenzymes for the sequence-specific cleavage of DNA (seefurther below) However REases with the desiredsequence-specific cleavage were soon isolated and theseset the stage for the advances in gene analysis and ma-nipulation collectively called lsquorecombinant DNA technol-ogyrsquo that quickly followed The first of these newenzymes HindII was discovered in Hamilton (lsquoHamrsquo)Smithrsquos laboratory at Johns Hopkins Medical School in1970 (29) This was subsequently termed a Type II REaseas its properties were distinct from the Type I REases (25)Purified from Haemophilus influenzae serotype d HindII(originally called endonuclease R) was found to act as ahomodimer and to cleave DNA at the symmetric (thoughdegenerate) sequence GTYrsquoRAC (Y=C or T R=A or

Grsquo indicates the cut site) (2930) Subsequently what wasthought to be pure HindII was found to be a mixture ofHindII and a second REase made by the same bacteriumHindIII HindIII cleaved DNA at a different symmetricsequence ArsquoAGCTT (3132) [see (33) for a thought-provoking discussion] The existence of HindIII came tolight during experiments to characterize the MTaseactivities of H influenzae These experiments showedthat the HindII and HindIII MTases acted at the sameDNA sequences as those cleaved by the REases Theymodified these sequences rather than cleaving themproducing GTYRm6AC and m6AAGCTT respectively(34ndash36)The universe of enzymes in this Type II category

expanded rapidly As Smithrsquos work proceeded on theeast coast of the USA REases with similar behaviourbut different specificity were discovered in the laboratoryof Herb Boyer at the University of California SanFrancisco on the west coast Here PhD student RobertYoshimori (37) benefited from the experience of DaisyDussoix who had moved from Werner Arberrsquos lab toUCSF Yoshimori investigated restriction systemspresent on plasmids in clinical E coli isolates andpurified what became known as EcoRI and EcoRII(3738) The EcoRI REase was found to cleave GrsquoAATTC (3940) and the corresponding MEcoRI MTase tomodify the inner adenines in this sequence producingGAm6ATTC (41) The EcoRII REase was found tocleave lsquoCCWGG (W=A or T) and the MEcoRIIMTase to modify the inner cytosines producingCm5CWGG (4243)

Staggered cuts and the advent of genetic engineering

In contrast to the Type I and Type III enzymes studied inthe 1960rsquos EcoRI and HindIII cleave DNA within theirrecognition sites and most importantly produce stag-gered cuts Since the recognition sites are symmetric thismeans that every fragment is flanked by the same single-stranded extension allowing any fragment to anneal (viathe extensions) to any other fragment thus setting thestage for recombining DNA fragments and lsquocloningrsquoThese findings were presented at the 1972 EMBOWorkshop on Restriction organized by Werner Arber(see Supplement S1 for details of the program and at-tendees) Figure 2 shows a photograph of participants atthis Workshop recalled by Noreen Murray as the mostexciting meeting in the history of the REases with discus-sions on the impact of this vital new information onlsquosticky endsrsquo and the implications for novel DNA manipu-lation The recently described DNA ligase (44) wouldallow the joining of DNA fragments with the samesticky ends EcoRI and HindIII spurred the developmentof recombinant DNA work through the availability ofboth purified enzymes and of replicatable carriers knownas vectors Both phage lambda (45) and various plasmids(4647) were developed into vectors into which DNA frag-ments generated by EcoRI and HindIII could be ligatedFittingly in 1978 Werner Arber was awarded the

Nobel Prize together with Dan Nathans and Ham Smith

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in recognition for their pioneering work on R-M (wwwnobelprizeorg)

Emerging genetic and enzymatic complexity

While the 1972 review by Matt Meselson et al (26)mentions only the recognition sequence of HindII thepace soon quickened The discovery of new restrictionenzymes skyrocketed as laborious in vivo phage-platingassays were replaced by rapid in vitro DNA-cleavageassays of cell extracts Elucidation of differences in recog-nition and cutting led to the classification of additionaldistinct classes or types of restriction enzymes (2548)which with extensions and subdivisions has stood thetest of time Type I (exemplified by EcoKI EcoBIEcoR124 the lsquoclassicalrsquo enzymes) Type II (EcoRIHindIII EcoRV the lsquoorthodoxrsquo enzymes) and Type III(EcoP1I and EcoP15I) Table 1 and Figure 1 Type IV(modification-dependent REases Mcr and Mrr) wasadded later (49) Sequencing and biochemistry havesince led to subdivisions within the Type I (see below)and Type II systems (Table 2) [see (4950) and httprebasenebcom for nomenclature and details]

The recombinant DNA scare

In their 1975 review (51) Nathans and Smith discussmethods for DNA cleavage and separation of the resultingfragments on gels as well as the use of REases inother applications eg the physical mapping of chromo-somes taking Simian Virus SV40 (SV40) as an example

The debate on the safety of recombinant DNA technologystarted soon after the 1972 EMBO Workshop and reportson the transfer of eukaryotic DNA into E coli [docu-mented by (52)] The debate was extremely heated butby 1990 many of the fears had abated as the anticipateddangers did not materialize and the advantages of DNAcloning and the ability to produce large quantities ofpharmaceutically important proteins such as insulinhormones and vaccines became clear

FURTHER HIGHLIGHTS IN THE STUDY OF TYPE IR-M SYSTEMS

Type I families are defined by complementation anddisplay sequence conservation

Type I REases were originally identified in E coli andother enteric organisms as barriers to DNA entry Theyturned out to be oligomeric proteins encoded by the threehost specificity determinant (hsd) genes a restriction (R)modification (M) and recognition (S for specificity) generespectively (Table 1 and Figure 1) Before the develop-ment of DNA sequencing genetic complementation testsdefined the hsdR hsdM and hsdS genes (5354) DNA hy-bridization studies and probing with antibodies directedat EcoKI established that EcoKI and EcoBI were moreclosely related to each other than to EcoAI the Type Isystem in E coli 15T [reviewed in (8)] This approachbased on biological interaction led to the division ofthese systems into families the Type IA (EcoKI EcoBI

Figure 2 Photograph of the participants at the EMBO Workshop on restriction in Leuenberg (Basel) Switzerland 26ndash30 September 1972 organizedby Werner Arber who took the picture (Archive Noreen Murray) Supplement S1 contains a list with names of the attendees and the program of thismeeting and puts names to faces as far as the attendees could be identified (from the archives of Noreen Murray and Werner Arber)

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EcoDI and Salmonella typhimurium StySPI) Type IB(EcoAI EcoEI and Citrobacter freundii CfrAI) Type IC(EcoR124 EcoDXXI EcoprrI) (8) and later Type ID(StyBLI and Klebsiella pneumoniae KpnAI) (95556) andType IE (KpnBI) (57) see reviews for further details (8ndash105859) Other organisms will have their own familiesfor example Staphylococcus aureus has at least twofamilies [(60) and unpublished DTFD results]

Preparation cofactor requirements and structures

Landmark studies on purified enzymes in the wake of the1962 Arber and Dussoix articles (1761) date to 1968 StuLinn and Werner Arber in Switzerland and MattMeselson and Bob Yuan in the USA respectivelypurified EcoBI and EcoKI They used restriction ofphages fd and lambda as their assay for detecting theenzymes during purification a laborious process (6263)This was no simple matter Bob Yuan recalls that lsquothe fallflew by in deep frustrationrsquo until he and Matt discoveredthat the enzyme needed S-adenosylmethionine (SAM) foractivity in addition to Mg2+and ATP (See Supplement S2for his personal story) The same cofactor requirementwas also found for EcoBI (6264) reviewed in (2526)Twenty-five years later we have come to appreciate thatSAM like ATP is a widely used cofactor in many meta-bolic reactions (6566)

A long-awaited breakthrough did not happen untilmuch later The structures of the subunits and assembledType I R-M enzymes Two structures of S subunitsappeared in 2005 and culminated in 2012 with the struc-ture of two complete R-M enzymes containing two Rsubunits two M subunits and one S subunit (67ndash69)

Type I enzymes cut away from the target site

In 1972 Horiuchi and Zinder showed that the DNArecognition site of EcoBI is not the cleavage site (70)

They cut 3H-labelled double-strand RF DNA of phagef1 (a relative of phage M13) with EcoBI denatured andrenatured the DNA and then treated with EcoBI a secondtime This resulted in a heterogeneous distribution ofsmall DNA fragments on alkaline sucrose gradientsleading to the conclusion that EcoBI cuts at a variabledistance from its target site This feature is now knownto be common to all Type I restriction enzymes It waslater shown by Studier that EcoKI would preferentiallycleave DNA approximately half-way between consecutivetarget sites (71) This feature is also common to all Type Irestriction enzymes although the distribution of cleavagelocations can be broad

DNA translocation to reach the cutting sites usesmolecular motors

Translocation was first observed in electron microscope(EM) studies that showed DNA looping by EcoBI andEcoKI These were interpreted as reaction intermediatesformed by the enzymes translocating along the DNAwhile remaining attached to their recognition sites(7273) In the case of EcoKI studies with full-lengthphage lambda DNA and relaxed or supercoiled circularDNA showed that EcoKI translocates the DNA pastitself concomitant with a large conformational changeof the enzyme creating large bidirectional loops clearlyvisible in the EM Recent studies confirm that on DNAbinding the enzyme strongly contracts from an open to acompact form (586974) In contrast EcoBI appeared toform loops in only one direction Later studies with EcoBIdid show supercoiled structures like EcoKI howevertranslocation was still unidirectional and without anyapparent strand selectivity in the cleavage reaction(7576) The translocation process would explain thecleavage observed half-way between consecutive targetsites on a DNA molecule as two translocating enzyme

Table 2 Nomenclature of Type II restriction enzymes

Subtype Features of restriction enzymesa Examples

Type IIP Palindromic recognition sequence recognized by both homodimeric and monomericenzymes cleavage occurs symmetrically usually within the recognition sequence

Prototypes EcoRI amp EcoRV

Type IIA Asymmetric recognition sequence FokIType IIB Cleavage on both sides of the recognition sequence BcgIType IIC Single combination R-M polypeptide HaeIVType IIE Two sequences required for cleavage one serving as allosteric effector EcoRII Sau3AIType IIF Two sequences required for cleavage concerted reaction by homotetramer SfiIType IIG Requires AdoMet cofactor for both R-M Eco57IType IIH Separate M and S subunits MTase organization similar to Type I systems BcgIType IIM Require methylated recognition sequence Type IIP or Type IIA DpnIType IIS Asymmetric recognition sequence cleavage at fixed positions usually outside recogni-

tion sequenceFokI

Type IIT Heterodimeric restriction enzyme Bpu10I BslIPutatives All subtypesControl Control proteins of Type II restriction enzymes CBamHI CPvuII

The characteristics of the orthodox Type IIP enzymes originally distinguished this group of enzymes from the Type I and III R-M systems Type IIPis the largest group owing to its valuable role in molecular science and its commercial value but the current classification and growing number ofR-M systems (putatively) identified makes it clear that Type II enzymes are highly diverse and the boundaries with the other types are beginning toblur see also Figure 3 and text for detailsaThese classifications reflect enzyme properties and activities and not their evolutionary relationships The classifications are not exclusive and oneenzyme can often belong several classes Thus BcgI for example is Type IIA B C G and H (see text for details)

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molecules would collide roughly half-way between targetsitesThe R-subunit of Type I enzymes belongs to the SNF2

helicasetranslocase superfamily of proteins These appearto be the result of an ancient fusion between nuclease andATP-dependent RecA-like (AAA+or lsquomotorrsquo) domains alinkage found in many enzymes involved in DNA repairreplication recombination and chromosome remodelling(77ndash88) As such Type I enzymes could prove useful forunderstanding the action of SNF2 enzymes in higherorganisms including the coordinated steps of DNAscanning recognition binding and alteration of thehelical structure that allow other domains or subunitsto move and touch the DNA All of these steps arerequired to prevent indiscriminate nuclease activity (69)The key to the functionality of the Type I REase and

other SNF2 proteins is their enormous flexibility allowinglarge conformational changes First noticed for EcoKI byYuan et al (73) and more recently for RecB and EcoR124(6989) large protein motions may be a general feature ofSNF2 proteins In line with such large-scale domainmovement mutational analysis of EcoR124 showedlong-range effects eg nuclease mutants affect thedistant helicase domain leading to a reduced translocationand ATP usage rate a decrease in the off rate slowerrestart and turnover In other words the nuclease andmotor domain together are lsquomore than the sum of theirpartsrsquo (8990)

Plasticity of type I DNA sequence recognition hybridspecificities and phase variation

Type I enzymes recognize bipartite DNA sequences [egAAC(N6)GTGC for EcoKI] The S subunit has aduplicated organization two 150 aa variable regionsalternate with smaller conserved regions which arehighly similar within each of the five families Eachvariable region recognizes one part of the bipartitetarget sequenceA key event in understanding the significance and mech-

anism of variation of sequence specificity was the discov-ery of a brand-new specificity resulting from a geneticcross (9192) As a result of crossing-over in the conservedcentral region between the two variable regions hybridspecificities were found This change in specificity wasfound to occur in vivo and in vitro and was first notedfor Salmonella species (91ndash94) An extensive treatment ofthis topic is found in the accompanying Type I reviewA variety of genetic processes promote variation in S

subunits proteins of Type I and Type III enzymes InLactococcus lactis entering plasmids may bring hsdSgenes with them (95) The segmented organizationof hsdS with two DNA recognition domains lendsitself to variation by DNA rearrangement Site-specificrecombination leads to expression of S proteins withalternative recognition domains in Mycoplasmapulmonis thus generating combinatorial variations of rec-ognition sequence (96) Such plasticity of restriction spe-cificity is also inferred in Bacteroides fragilis (97) and otherspecies

FURTHER HIGHLIGHTS IN THE STUDY OF TYPE IIR-M SYSTEMS

Subdivisions of type II enzymes

Type II REases are defined rather broadly as enzymesthat cleave DNA at a fixed position with respect to theirrecognition sequence and produce distinct DNA-fragment banding patterns during gel electrophoresisThese REases are extremely varied and occur in manystructural forms The Type II classification was used ori-ginally to define the simplest kind of REases exemplifiedby HindII and EcoRI that recognize symmetric DNAsequences and require Mg2+ ions for cleavage activity(25) Enzymes of this sort generally act as homodimersand cleave DNA within their recognition sequencesIn vivo they function in conjunction with a separatemodification MTase that acts independently as amonomer (Table 1) The first distinction made amongType II enzymes concerned REases such as HphI andFokI that recognize asymmetric sequences and cleave ashort distance away to one side These were designatedType IIS (98)

As the number of REases producing distinct frag-ments grew it became clear that many unrelatedproteins were included in the category (99) Rather thandividing these into further Types based on theirphylogenies it was agreed that lsquoType IIrsquo should becomea utilitarian classification that reflected enzymaticbehaviour rather than evolutionary relatedness and forconvenience a number of Type II groups correspondingto particular enzymatic behaviours were defined (49)(Table 2) Each of these groups A B C E F G HM P S and T should be thought of not as an exclusivesubdivision but rather as an icon that signifies somespecific property Enzymes may exhibit more than onesalient property and thus belong to more than onegroup HindIII and EcoRI remain simple they aremembers of just the one Type IIP group (lsquoPrsquo forPalindromic) BcgI in contrast is complicated since itrecognizes an asymmetric DNA sequence (= Type IIA)cleaves on both sides of that sequence (= Type IIB) andcomprises a fused endonuclease-methyltransferase subunit(= Type IIC) plus a Type I-like DNA-specificity subunit(= Type IIH) BcgI thus is a member of multiple groups(100ndash102) DpnI (Gm6ArsquoTC) is a Type IIM REase whichcleaves its recognition sequence only when the sequence ismethylated (103) DpnI is also a member of the Type IIPgroup since its recognition sequence is palindromicsequence and cleavage is internal and symmetric(Figure 3 and httprebasenebcom)

Type IIP (lsquoorthodoxrsquo) REases such as EcoRI andHindIII were crucial to the development of recombinantDNA technology Certain lsquounorthodoxrsquo enzymes havealso been widely used Sau3AI (rsquoGATC) is a monomericType IIE REase that dimerizes on the DNA inducingDNA loops Two recognition sites must be bound foractivity one is cleaved while the other acts as allostericeffector (104) EcoRII is somewhat similar and manyREases are now known to cleave only as dimers ofdimers bound to two separate sites

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Predicting enzyme families sequences structures andbioinformatics

Early amino acid sequences of Type II enzymes (egEcoRI EcoRV PvuII and BamHI) showed them to bealmost completely unrelated (105ndash109) When crystalstructures appeared (110ndash114) commonalities began toemerge The motif PD-(DE)XK was identified as acommon feature (115116) This motif also appeared inother nucleases eg lambda exonuclease (117) and theTn7 transposase protein TnsA (118) This motif is thecatalytic core of a Mg2+-dependent nuclease

REBASE the Restriction Enzyme Database set up byRich Roberts to keep track of RM specificities andindicate how to acquire the enzymes made possible thenext phase of understanding First on paper (119120)then via the nascent Internet by File Transfer Protocoland finally on the World Wide Web (1121) this resourcemakes available a focused organized data set allowingcomputational analysis of sequences and structures aswell as access to individual topics of interest [eg(99122ndash129)]

Recently Sau3A (104) and several other REases provedable to cut DNARNA hybrids (130) The rarity of thisproperty (6 of 223 surveyed) suggests that any biologicalroles for this ability will be specialized but the propertycould be used to study the ubiquitous small RNA mol-ecules that regulate expression in all domains of life (131)

FURTHER HIGHLIGHTS IN THE STUDY OF TYPE IIIR-M SYSTEMS

Type III enzymes have properties that are intermediatebetween Types I and II (Table 1 and Figure 1) Ingeneral Type III enzymes recognize asymmetric

sequences cleave 25ndash27 nucleotides away from their rec-ognition site and use ATP and SAM as cofactorsalthough they do not have an absolute requirement forthe latter Particularly interesting topics include controlof the phage-borne REcoP1I REase activity following in-fection and how newly replicated DNA can be protectedwhen only one strand of the recognition sequence is pro-tected by methylation (132ndash139)An early result showed that two copies of the target site

were required for DNA cleavage but that these sites had tobe in a head-to-head orientation (135140) A head-to-tailorientation prevented cleavage How this communicationbetween the two target sites was achieved when ATP hy-drolysis was insufficient for DNA translocation like theType I enzymes (59) has provoked much discussion(141) It appears that DNA looping may have a role inbringing the sites together (142143) but recent single-molecule analyses (144145) show strong evidence forenzyme diffusion along the DNA triggered by an ATP-dependent conformational change as a novel mechanismfor bringing two copies of the enzyme together to givecleavage see also (83146) The long-awaited atomic struc-ture of a Type III R-M enzyme should resolve many of thecomplexities of these enzymes [AK Aggarwal personalcommunication (147148)]

FURTHER HIGHLIGHTS IN THE STUDY OF TYPE IVRESTRICTION SYSTEMS

Modification-dependent restriction was first observedwith populations of phage T4 that containedhydroxymethylcytosine (hm5C)-substituted DNA (13)reviewed in (149150) This original discovery relied onthe fortuitous use of Shigella dysenteria SH as permissivehost it lacks both of the E coliK-12 hm5C-targeted endo-nucleases and also the donor for the protective modifica-tion glucosylation This allowed glucoseless phage to bepropagated in Shigella while picking apart the E coliK-12 set of restricting and modifying genesKey advances in the early years lay in determining the

nature of the modifications in T-even phage DNA and thegenes that enable them hm5C is incorporated intothe DNA during synthesis and then glucose residues areadded in different configurations The host provides theglucosyl donor (151152) while the phage provides theglucosyltransferase enzymes (153ndash155) With thesegenetic tools in hand the host genes mediating thephage restriction activity were identified (156) Thesewere named rglA and rglB (restricts glucoseless phage)because they mediate restriction of hmC-containingphage that lack the further glucose modificationIn the 1980s the focus switched to other modifications

particularly m5C with efforts to clone Type II MTasesand eukaryotic DNA into E coli (157ndash159) The m5C-specific functions mcrA and mcrB were mapped (160)and were shown to be identical to the rglA and rglBgenes (161) A third modificationndashdependent enzyme wasfound to recognize m6A as well as m5C (162) Using thegenetic tools described above glucose-specific activity wasidentified (163164) Most recently a newly described

Type II restriction enzymes grouped by cleavage properties

Type IIP homodimer Type IIS Type IIB

Type IIE Type IIF

Type II monomer (eg BcnI)

Cleave detach rotatereattach

Figure 3 Type II restriction enzymes grouped by cleavage propertieslsquoOrthodoxrsquoIIP enzymes (eg EcoRI EcoRV) cut at the recognition siteType IIS cut away from the site (eg FokI BfiI) Type IIB require tworecognition sites and cut on the outside (eg BplI) Type IIE requiretwo recognition sites and one of the two sites acts as allosteric effector(eg EcoRII) Type IIF require two sites and cut at both sites as atetramer after bringing the two regions together by looping the DNA(eg SfiI) Enzymes such as BcnI act as a monomer in contrast to mostType II REases that act as dimers See Table 2 and text for furtherdetails

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DNA modification (165) has provided new targets (166)for Type IV enzymes phosphorothioate linkages in thephosphodiester backboneThe utility of all these discoveries was at first the ability

to avoid them (167ndash169) these restriction systems werefound to underlie difficulties encountered in the introduc-tion of foreign MTases into E coli (157158170) On thepositive side use of Type IV restriction in vivo alsoallowed enrichment of clone libraries for active eukaryoticgenomic sequence since much transcriptionally silencedDNA is heavily methylated [eg (171)]Type IV enzymes have aroused considerable interest in

recent years following the rediscovery of hm5C in theDNA of higher eukaryotes (172ndash175) This finding couldportend the discovery of further as yet unknown or neg-lected DNA modifications The ability of Type IVenzymes to distinguish between C m5C hm5C andother molecular variations of cytosine implicates theseenzymes as useful tools for studies of epigenetic phenom-ena the commercially available enzyme McrBC has beenused for the study of such modification patterns (176177)Much history may remain to be written The accom-

panying review focuses on structural and enzymaticproperties of the systems that are known and sketchessome of the evolutionary pressures faced by restrictionsystems as they compete with each other and withinvading replicons

CONTROL OF RESTRICTION

Double-stranded cleavage of cellular DNA is extremelydeleterious to the host cell even when it can be repairedEarly in the study of restriction systems the ease ofmoving systems among strains with differing systems byconjugation or transduction was noted This suggestedthat regulation must be present to enable exchange ofactivities More recently the sporadic distribution ofR-M systems in genomes of closely related strainsstrongly suggests that acquisition of a new system is arelatively frequent event in nature as well Thus coordin-ation of expression or activity of the R-M activities is akey research topic Transcriptional or translationalcontrol of Type I systems has not been documenteddespite efforts to find it (8178) However post-transla-tional control is exerted at several levels and is describedin the accompanying review on Type I R-M systems Thecontrol of Type II R-M systems recapitulates the mechan-isms for other regulatory systems and is described here

Transcriptional control of Type II enzyme expression

In contrast to Type I enzymes transcriptional control hasbeen found for Type II enzymes Most of the Type IIsystems that have been examined have the problem ofintegrating control of the modification and restrictionactivities separately since they are embodied in separateproteins Once again the introduction of these genes intoa naıve host is of special interest

Control of restriction of Type II enzymes the caseof EcoRIExpression of the MTase gene and methylation of the hostDNA before synthesis of the REase is the obvious solutionand the so-called lsquoHungarian trickrsquo was the basis for thecloning of many of the first restriction enzymes (179) Thelab of Ichizo Kobayashi investigated the regulation of theEcoRI gene ecoRIR (180ndash182) This gene is upstream ofthe modification gene ecoRIM The M gene has its ownpromoters embedded in ecoRIR and no transcription ter-minator between the genes so ecoRIM can be transcribedwith and without ecoRIR Using primer extension tolocate the start sites and gene fusions to assess expressiontwo adjacent promoters for ecoRIM as well as two reversepromoters were found within ecoRIR These convergentpromoters negatively affect each other [as in lambda(183)] Transcription from the reverse promoter isterminated by the forward promoters and generates asmall antisense RNA The presence of the antisenseRNA gene in trans reduced lethality mediated bycleavage of under-methylated chromosomes after loss ofthe EcoRI plasmid (post-segregational killing) (182184)

Dual transcription control by C proteinsThe Blumenthal laboratory provided the first evidence fortemporal control in the plasmid-based PvuII system ofProteus vulgaris (108185) A similar open reading framewith similar function was also found contemporaneouslyin the BamHI system (186187) In the PvuII system theMTase is expressed without delay from an independentpromoter and protects the host DNA The REase geneis in an operon with that for an autogenous activatorre-pressor protein CPvuII Low basal expression from thepvuIIC promoter leads to accumulation of the activatorthereby boosting transcription of the C and REase genes(108185) (Figure 4)

The C protein binds to palindromic DNA sequences(C boxes) defining two sites upstream of its gene OLassociated with activation and OR associated with repres-sion The C protein activates expression of its own gene aswell as that of the REase (188) The regulation is similar togene control in phage lambda differential bindingaffinities for the promoters in turn depend on differentialDNA sequence and dual symmetry recognition Cproteins belong to the helix-turn-helix family of transcrip-tional regulators that include the cI and cro repressorproteins of lambdoid phages

In the wake of PvuII and BamHI other R-M systemswere discovered that were controlled by C proteinsincluding BglII (189) Eco72I (190) EcoRV (191)Esp396I (192) and SmaI (193) Currently Rebase lists 19documented C proteins as well as 432 putatives based onsequence data (16 September 2013 httprebasenebcom) The organization of the genes in the system andregulatory details differ from system to system(108185194) There is no published evidence addressingthe question of whether R-M systems as a whole evolve inconcert with the C proteins An interesting system wouldbe one homologous to a C-regulated system but withoutthe C gene

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Structure of C proteinsThe first structures of C-proteins appeared in 2005CAhdI from Geoff Knealersquos laboratory (195) andCBclI from a consortium of workers (196) The structureswere solved without bound DNA and while they con-firmed the close relationship between C-proteins andhelix-turn-helix DNA-binding proteins in general theydid not reveal details of the interactions betweenC-proteins and their C-box binding sites in DNA(195197ndash204) That came 4 years later with the crystalstructure of CEsp1396I bound to DNA (205) This struc-ture coupled with experimental investigations revealedthe mechanics of the genetic switch and the nature ofthe sequence-specific and non-specific interactions withthe promoters controlling the CR and M genes (205ndash208) CEsp1396I bound as a tetramer with two dimersbound adjacently on the 35-bp operator sequence OL+OR

(206) This cooperative binding of dimers to the DNAoperator controls the switch from activation to repressionof the C and R genes

Biological consequences of transcriptional regulationThe existence of C proteins explains why it was difficult tointroduce some R-M genes in E coli For instance theBamHI system of Bacillus amyloliquefaciens could only

be maintained in E coli when the REase and MTasewere present on one plasmid with an additional copy ofthe MTase on a second plasmid (209) Further analysissuggested that in Bacillus subtilis a host more closelyrelated to the original expression of R-M was even morestringently regulated (109) CBamHI enhanced activity ofthe REase 100-fold in E coli but at least 1000-fold inB subtilis In E coli the C protein repressed expressionof the MTase 15-fold The B subtilis vegetative RNApolymerase is known be more stringent in its promotersequence requirements than that of E coli (210)possibly accounting for the difference in behaviour inthe two speciesCrosstalk among the C genes of similar specificity can

allow exclusion with R-M systems of different sequencespecificities because of the premature activation of the Rgene The pvuIIC and bamHIC genes define one incom-patibility group of exclusion whereas ecoRVC definesanother (211) Entry of a second R-M system thusbecomes lethal a phenomenon called lsquoapoptotic mutualexclusionrsquo (211)

THE IMPACT OF RESTRICTION ENZYMES

The technical ingenuity applied to the use of restrictionenzymes warrants a separate detailed Survey andSummary or indeed an entire book For instance theiruse led to the production of insulin from recombinantbacteria and yeast by Genentech thus greatly increasingthe supply for diabetics and the production of a recom-binant vaccine for Hepatitis B by Biogen to treat thehundreds of millions of people at risk of infection bythis virus More recently they have been redesigned tocreate artifical nucleases the Zinc-finger nucleases andthe TAL-effector nucleases which have potential forgene targeting and gene therapy (212) Here we limit our-selves to a few other examples with significant scientific orpublic impact

Genetic engineering

Type II enzymes yielded many practical benefits as E coliK12 its genes and its vectors became the workhorses ofmolecular biology in the 1970s for cloning generation oflibraries DNA sequencing detection and overproductionof enzymes hormones etc [eg (45213ndash224)] The appli-cations of Type II enzymes continued to expand espe-cially after the arrival of synthetic DNA in vitropackaging of DNA in phage particles and improved bac-terial hosts and vectors for overexpression and stabiliza-tion of proteins [see eg (225ndash232)]A historical perspective on the above topics is beyond

the scope of this review However a couple of vignettesillustrate how use of REases enabled the research commu-nity to leverage a store of understanding to create tools fornew advancesThe lacZ gene which had EcoRI sites suitable for early

vectors and its encoded enzyme beta-galactosidase had along history of investigation Its utility in the creation ofcloning vectors relied on identification of domains withinthe encoded protein namely a large catalytic domain and

The PvuII and Esp1396I operons

M RC

pmod 1 2

pres

pres 1

MRC

pres

pres 1

OmOROL

pmod

OROL PvuII

Esp1396I

Figure 4 Intricate control of restriction in the operons of the Type IIR-M systems of PvuII and Esp1396I by controlling C proteins A smallC gene upstream of and partially overlapping with R is coexpressedfrom pres1 located within the M gene at low level with R after entry ofthe self-transmissible PvuII plasmid into a new host while M is ex-pressed at normal levels from its own two promoters pmod1 and pmod2

located within the C gene A similar C protein operates in Esp1396Ibut in this case the genes are convergently transcribed with transcrip-tion terminator structures in between and M is expressed from apromoter under negative control of operator OR when engaged by Cprotein in a manner similar to that of the PvuII system Briefly the Cprotein binds to two palindromic sequences (C boxes) defining operatorsites OR and OL upstream of the C and R genes After initial low-levelexpression of CPvuII protein from the weak promoter pres1 positivefeedback by high-affinity binding of a C protein dimer to the distal OL

site later stimulates expression from the second promoter pres resultingin a leaderless transcript and more C and R protein The proximal siteOR is a much weaker binding site but C protein bound at OL enhancesthe affinity of OR for C protein and at high levels of C protein theprotein-OR complex downregulates expression of C and R In this wayC protein is both an activator and negative regulator of its own tran-scription In addition it is a negative regulator of M which makessense as overmethylation of DNA may also be harmful to the cell(see text for further details) CEsp1396I controls OR OL and OM ina similar manner as described above In this way C proteins keep bothR-M under control and have been tentatively identified in gt300 R-Msystems (Table 2)

Nucleic Acids Research 2014 Vol 42 No 1 11

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a small multimerization domain This was discovered bythe Muller-Hill group in 1974 (233234) The 25N-terminal residues of the small domain can be replacedby peptides of any size and origin without destroying theability of the multimerization domain to interact with thecatalytic domain (233234) As a result vectors with shortstretches of DNA carrying multiple restriction sites couldbe created (235ndash237) Cloning into these sites interruptedthe translation of the small domain destroying its abilityto interact with the separately expressed large one Thismade possible rapid screening of bacterial colonies on anagar plate for those lacking the activity of LacZ using acolour assayIn addition vectors carrying the intact gene but with

multiple cloning sites allowed EcoRI-based DNA con-structs for transcriptional and translational fusions tothe lacZ gene (238ndash250) The majority (90) of suchLacZ-fusion proteins are stable allowing purification ofchimeric antigens as well as detection of positive cloneswith colour assays (238251) Mutagenesis studies in thelaboratory of Jeffrey Miller used the lacZ gene in phagef1 allowing the rapid detection of spontaneous or inducedbase substitutions and frameshifts (252ndash254) This resultedin eg LacZ-transgenic mice for studies on DNA damagein different organs and tissues in mammalian cells(255256)

DNA fingerprinting

Restriction enzymes are tools for monitoring RestrictionFragment Length Polymorphisms allowing the locationof mutations generation of human linkage maps identi-fication of disease genes (such as sickle cell trait orHuntington disease) and last but not least the DNAfingerprinting technique developed by Alec Jeffreys(257ndash267) DNA fingerprinting (268) allows the solutionof paternity cases the identification of criminals and theirvictims and the exoneration of the falsely accused The useof REases in this system enabled the creation of suitableprocedures for such identification although PCR haslargely displaced REases in this applicationREases have also proven useful for identifying patho-

genic bacterial strains most recently of S aureus sp withantibiotic-resistance and virulence factors mediated bymobile genetic elements eg the methicillin-resistantS aureus (MRSA) bacteria (269) Such strains pose agreat threat to humans and animals (270)

FINAL THOUGHTS

In 1977 Werner Arber proposed that REases might haveadditional functions in the cell (271) and this is an idea tokeep in mind given that much of the study of restrictionenzymes has been aimed at creating tools rather than abasic study of their behaviour in their natural hostsFor example the actions of translocating enzymes such

as the Type I and IV enzymes at a replication fork or othervariant structure are one such possibility (272273) Thisactivity may seem of arcane interest but a broader under-standing especially of the translocating enzymes couldfurther understanding of genome stabilization activities

in all domains of life Applications to genome manipula-tion or medicine could emerge Action at aberrant struc-tures is a major topic of interest in medicine (274)

Lastly it is interesting to speculate on the condition ofmolecular biology and all of its associated sciences at thepresent day if the simple experiment of spreading bacteriaand phage on agar plates to follow the restriction-modifi-cation phenomenon (13ndash15) had not been pursued It isclear that a multi-billion dollar biotechnology industrywould not have been spawned medical diagnostics andthe treatment of many diseases would have been severelyretarded genomics and genome sequencing projects wouldhave been difficult if not impossible and their support ofbioinformatics and evolutionary studies would also nothave been possible thus greatly diminishing our currentappreciation of the spectacular diversity of life on earth

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

We thank the many colleagues who generously providedinformation for this review Attendees of the 1972 EMBOWorkshop helped identify the people in Figure 2 Specialthanks to Werner Arber Joe Bertani and Rich Roberts forhistorical material reprints from their archives andhelpful emails over the past decade

FUNDING

Funding for open access charge New England Biolabs

Conflict of interest statement None declared

REFERENCES

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2 RobertsRJ and ChengX (1998) Base flipping Annu RevBiochem 67 181ndash198

3 HortonJR LiebertK BekesM JeltschA and ChengX(2006) Structure and substrate recognition of the Escherichia coliDNA adenine methyltransferase J Mol Biol 358 559ndash570

4 ChengX and BlumenthalRM (2002) Cytosines do it thyminesdo it even pseudouridines do itmdashbase flipping by an enzyme thatacts on RNA Structure 10 127ndash129

5 YangW (2011) Surviving the sun repair and bypass of DNAUV lesions Protein Sci 20 1781ndash1789

6 BrooksSC AdhikaryS RubinsonEH and EichmanBF(2013) Recent advances in the structural mechanisms of DNAglycosylases Biochim Biophys Acta 1834 247ndash271

7 TubbsJL PeggAE and TainerJA (2007) DNA bindingnucleotide flipping and the helix-turn-helix motif in base repairby O6-alkylguanine-DNA alkyltransferase and its implications forcancer chemotherapy DNA Repair (Amst) 6 1100ndash1115

8 MurrayNE (2000) Type I restriction systems sophisticatedmolecular machines (a legacy of Bertani and Weigle) MicrobiolMol Biol Rev 64 412ndash434

9 MurrayNE (2002) 2001 Fred Griffith review lectureImmigration control of DNA in bacteria self versus non-selfMicrobiology 148 3ndash20

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10 LoenenWA (2003) Tracking EcoKI and DNA fifty years on agolden story full of surprises Nucleic Acids Res 31 7059ndash7069

11 PingoudA and JeltschA (2001) Structure and function of typeII restriction endonucleases Nucleic Acids Res 29 3705ndash3727

12 PingoudA FuxreiterM PingoudV and WendeW (2005) TypeII restriction endonucleases structure and mechanism Cell MolLife Sci 62 685ndash707

13 LuriaSE and HumanML (1952) A nonhereditary host-inducedvariation of bacterial viruses J Bacteriol 64 557ndash569

14 AndersonES and FelixA (1952) Variation in Vi-phage II ofSalmonella typhi Nature 170 492ndash494

15 BertaniG and WeigleJJ (1953) Host controlled variation inbacterial viruses J Bacteriol 65 113ndash121

16 LuriaSE (1953) Host-induced modifications of viruses ColdSpring Harb Symp Quant Biol 18 237ndash244

17 ArberW and DussoixD (1962) Host specificity of DNAproduced by Escherichia coli I Host controlled modification ofbacteriophage lambda J Mol Biol 5 18ndash36

18 ArberW HattmanS and DussoixD (1963) On the host-controlled modification of bacteriophage lambda Virology 2130ndash35

19 GloverW SchellJ SymondsN and StaceyKA (1963) Thecontrol of host-induced modification by phage P1 Genet Res 4480ndash482

20 LederbergS (1966) Genetics of host-controlled restriction andmodification of deoxyribonucleic acid in Escherichia coliJ Bacteriol 91 1029ndash1036

21 FranklinNC and DoveWF (1969) Genetic evidence forrestriction targets in the DNA of phages lambda and phi 80Genet Res 14 151ndash157

22 ArberW (1965) Host-controlled modification of bacteriophageAnnu Rev Microbiol 19 365ndash378

23 ArberW and LinnS (1969) DNA modification and restrictionAnnu Rev Biochem 38 467ndash500

24 ArberW (1971) Host-Controlled Variation In HersheyAD(ed) Bacteriophage Lambda Cold Spring Harbor LaboratoryNew York pp 83ndash96

25 BoyerHW (1971) DNA restriction and modification mechanismsin bacteria Annu Rev Microbiol 25 153ndash176

26 MeselsonM YuanR and HeywoodJ (1972) Restriction andmodification of DNA Annu Rev Biochem 41 447ndash466

27 ArberW (1965) Host specificit of DNA produced by Escherichiacoli V The role of methionine in the production of hostspecificity J Mol Biol 11 247ndash256

28 GoldM HurwitzJ and AndersM (1963) The enzymaticmethylation of RNA and DNA II On the species specificity ofthe methylation enzymes Proc Natl Acad Sci USA 50164ndash169

29 KellyTJ Jr and SmithHO (1970) A restriction enzyme fromHemophilus influenzae II J Mol Biol 51 393ndash409

30 SmithHO and WilcoxKW (1970) A restriction enzyme fromHemophilus influenzae I Purification and general propertiesJ Mol Biol 51 379ndash391

31 LandyA RuedisueliE RobinsonL FoellerC and RossW(1974) Digestion of deoxyribonucleic acids from bacteriophageT7 lambda and phi 80h with site-specific nucleases fromHemophilus influenzae strain Rc and strain Rd Biochemistry 132134ndash2142

32 OldR MurrayK and BoizesG (1975) Recognition sequence ofrestriction endonuclease III from Hemophilus influenzae J MolBiol 92 331ndash339

33 HalfordSE (2009) An end to 40 years of mistakes in DNA-protein association kinetics Biochem Soc Trans 37 343ndash348

34 RoyPH and SmithHO (1973) DNA methylases of Hemophilusinfluenzae Rd I Purification and properties J Mol Biol 81427ndash444

35 RoyPH and SmithHO (1973) DNA methylases of Hemophilusinfluenzae Rd II Partial recognition site base sequences J MolBiol 81 445ndash459

36 RoszczykE and GoodgalS (1975) Methylase activities fromHaemophilus influenzae that protect Haemophilus parainfluenzaetransforming deoxyribonucleic acid from inactivation byHaemophilus influenzae endonuclease R J Bacteriol 123287ndash293

37 YoshimoriR (1971) A genetic and biochemical analysis of therestriction and modification of DNA by resistance transferfactors 146 p PhD Thesis University of California SanFrancisco ThesisDissertation

38 YoshimoriR Roulland-DussoixD and BoyerHW (1972) Rfactor-controlled restriction and modification of deoxyribonucleicacid restriction mutants J Bacteriol 112 1275ndash1279

39 HedgpethJ GoodmanHM and BoyerHW (1972) DNAnucleotide sequence restricted by the RI endonuclease Proc NatlAcad Sci USA 69 3448ndash3452

40 MertzJE and DavisRW (1972) Cleavage of DNA by R 1restriction endonuclease generates cohesive ends Proc Natl AcadSci USA 69 3370ndash3374

41 DugaiczykA HedgpethJ BoyerHW and GoodmanHM(1974) Physical identity of the SV40 deoxyribonucleic acidsequence recognized by the Eco RI restriction endonuclease andmodification methylase Biochemistry 13 503ndash512

42 BoyerHW ChowLT DugaiczykA HedgpethJ andGoodmanHM (1973) DNA substrate site for the EcoRIIrestriction endonuclease and modification methylase Nat NewBiol 244 40ndash43

43 BiggerCH MurrayK and MurrayNE (1973) Recognitionsequence of a restriction enzyme Nat New Biol 244 7ndash10

44 LehmanIR (1974) DNA ligase structure mechanism andfunction Science 186 790ndash797

45 MurrayNE and MurrayK (1974) Manipulation of restrictiontargets in phage lambda to form receptor chromosomes for DNAfragments Nature 251 476ndash481

46 CohenSN ChangAC BoyerHW and HellingRB (1973)Construction of biologically functional bacterial plasmids in vitroProc Natl Acad Sci USA 70 3240ndash3244

47 HershfieldV BoyerHW YanofskyC LovettMA andHelinskiDR (1974) Plasmid ColEl as a molecular vehicle forcloning and amplification of DNA Proc Natl Acad Sci USA71 3455ndash3459

48 SmithHO and NathansD (1973) Letter a suggestednomenclature for bacterial host modification and restrictionsystems and their enzymes J Mol Biol 81 419ndash423

49 RobertsRJ BelfortM BestorT BhagwatAS BickleTABitinaiteJ BlumenthalRM DegtyarevSK DrydenDTDybvigK et al (2003) A nomenclature for restriction enzymesDNA methyltransferases homing endonucleases and their genesNucleic Acids Res 31 1805ndash1812

50 TockMR and DrydenDT (2005) The biology of restrictionand anti-restriction Curr Opin Microbiol 8 466ndash472

51 NathansD and SmithHO (1975) Restriction endonucleases inthe analysis and restructuring of DNA molecules Annu RevBiochem 44 273ndash293

52 WatsonJD and ToozeJ (1981) The DNA Story DocumentaryHistory of Gene Cloning WH Freeman and Company SanFrancisco

53 BoyerHW and Roulland-DussoixD (1969) A complementationanalysis of the restriction and modification of DNA inEscherichia coli J Mol Biol 41 459ndash472

54 HubacekJ and GloverSW (1970) Complementation analysis oftemperature-sensitive host specificity mutations in Escherichia coliJ Mol Biol 50 111ndash127

55 TitheradgeAJ KingJ RyuJ and MurrayNE (2001) Familiesof restriction enzymes an analysis prompted by molecular andgenetic data for type ID restriction and modification systemsNucleic Acids Res 29 4195ndash4205

56 KasarjianJK HidakaM HoriuchiT IidaM and RyuJ(2004) The recognition and modification sites for the bacterial

type I restriction systems KpnAI StySEAI StySENI and StySGI

Nucleic Acids Res 32 e8257 ChinV ValinluckV MagakiS and RyuJ (2004) KpnBI is the

prototype of a new family (IE) of bacterial type I restriction-

modification system Nucleic Acids Res 32 e13858 DrydenDT MurrayNE and RaoDN (2001) Nucleoside

triphosphate-dependent restriction enzymes Nucleic Acids Res29 3728ndash3741

59 BourniquelAA and BickleTA (2002) Complex restrictionenzymes NTP-driven molecular motors Biochimie 84 1047ndash1059

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60 RobertsGA HoustonPJ WhiteJH ChenKStephanouAS CooperLP DrydenDT and LindsayJA(2013) Impact of target site distribution for Type I restrictionenzymes on the evolution of methicillin-resistant Staphylococcusaureus (MRSA) populations Nucleic Acids Res 41 7472ndash7484

61 DussoixD and ArberW (1962) Host specificity of DNAproduced by Escherichia coli II Control over acceptance ofDNA from infecting phage lambda J Mol Biol 5 37ndash49

62 LinnS and ArberW (1968) Host specificity of DNA producedby Escherichia coli X In vitro restriction of phage fd replicativeform Proc Natl Acad Sci USA 59 1300ndash1306

63 MeselsonM and YuanR (1968) DNA restriction enzyme fromE coli Nature 217 1110ndash1114

64 Roulland-DussoixD and BoyerHW (1969) The Escherichia coliB restriction endonuclease Biochim Biophys Acta 195 219ndash229

65 LoenenWA (2006) S-adenosylmethionine jack of all trades andmaster of everything Biochem Soc Trans 34 330ndash333

66 LoenenWAM (2010) S-adenosylmethionine simple agent ofmethylation and secret to aging and metabolismIn TollefsbolTO (ed) Epigenetics of Aging Springerpp 107ndash131

67 KimJS DeGiovanniA JancarikJ AdamsPD YokotaHKimR and KimSH (2005) Crystal structure of DNA sequencespecificity subunit of a type I restriction-modification enzyme andits functional implications Proc Natl Acad Sci USA 1023248ndash3253

68 CalistoBM PichOQ PinolJ FitaI QuerolE andCarpenaX (2005) Crystal structure of a putative type Irestriction-modification S subunit from Mycoplasma genitaliumJ Mol Biol 351 749ndash762

69 KennawayCK TaylorJE SongCF PotrzebowskiWNicholsonW WhiteJH SwiderskaA Obarska-KosinskaACallowP CooperLP et al (2012) Structure and operation ofthe DNA-translocating type I DNA restriction enzymes GenesDev 26 92ndash104

70 HoriuchiK and ZinderND (1972) Cleavage of bacteriophage flDNA by the restriction enzyme of Escherichia coli B Proc NatlAcad Sci USA 69 3220ndash3224

71 StudierFW and BandyopadhyayPK (1988) Model for howtype I restriction enzymes select cleavage sites in DNA ProcNatl Acad Sci USA 85 4677ndash4681

72 RosamondJ EndlichB and LinnS (1979) Electron microscopicstudies of the mechanism of action of the restriction endonucleaseof Escherichia coli B J Mol Biol 129 619ndash635

73 YuanR HamiltonDL and BurckhardtJ (1980) DNAtranslocation by the restriction enzyme from E coli K Cell 20237ndash244

74 KennawayCK Obarska-KosinskaA WhiteJH TuszynskaICooperLP BujnickiJM TrinickJ and DrydenDT (2009)The structure of MEcoKI Type I DNA methyltransferase with aDNA mimic antirestriction protein Nucleic Acids Res 37762ndash770

75 EndlichB and LinnS (1985) The DNA restriction endonucleaseof Escherichia coli B II Further studies of the structureof DNA intermediates and products J Biol Chem 2605729ndash5738

76 EndlichB and LinnS (1985) The DNA restriction endonucleaseof Escherichia coli B I Studies of the DNA translocation andthe ATPase activities J Biol Chem 260 5720ndash5728

77 DaviesGP MartinI SturrockSS CronshawA MurrayNEand DrydenDT (1999) On the structure and operation of typeI DNA restriction enzymes J Mol Biol 290 565ndash579

78 Fairman-WilliamsME GuentherUP and JankowskyE (2010)SF1 and SF2 helicases family matters Curr Opin Struct Biol20 313ndash324

79 GorbalenyaAE and KooninEV (1991) Endonuclease (R)subunits of type-I and type-III restriction-modification enzymescontain a helicase-like domain FEBS Lett 291 277ndash281

80 GorbalenyaAE KooninEV DonchenkoAP and BlinovVM(1988) A conserved NTPndashmotif in putative helicases Nature 33322

81 HallMC and MatsonSW (1999) Helicase motifs theengine that powers DNA unwinding Mol Microbiol 34867ndash877

82 SingletonMR DillinghamMS and WigleyDB (2007)Structure and mechanism of helicases and nucleic acidtranslocases Annu Rev Biochem 76 23ndash50

83 SzczelkunMD FriedhoffP and SeidelR (2010) Maintaining asense of direction during long-range communication on DNABiochem Soc Trans 38 404ndash409

84 TutejaN and TutejaR (2004) Prokaryotic and eukaryoticDNA helicases Essential molecular motor proteins for cellularmachinery Eur J Biochem 271 1835ndash1848

85 UmateP TutejaN and TutejaR (2011) Genome-widecomprehensive analysis of human helicases Commun IntegrBiol 4 118ndash137

86 RamanathanA and AgarwalPK (2011) Evolutionarilyconserved linkage between enzyme fold flexibility and catalysisPLoS Biol 9 e1001193

87 MahdiAA BriggsGS SharplesGJ WenQ and LloydRG(2003) A model for dsDNA translocation revealed by astructural motif common to RecG and Mfd proteins EMBO J22 724ndash734

88 RudolphCJ UptonAL BriggsGS and LloydRG (2010) IsRecG a general guardian of the bacterial genome DNA Repair(Amst) 9 210ndash223

89 SisakovaE StanleyLK WeiserovaM and SzczelkunMD(2008) A RecB-family nuclease motif in the Type Irestriction endonuclease EcoR124I Nucleic Acids Res 363939ndash3949

90 SisakovaE WeiserovaM DekkerC SeidelR andSzczelkunMD (2008) The interrelationship of helicase andnuclease domains during DNA translocation by the molecularmotor EcoR124I J Mol Biol 384 1273ndash1286

91 BullasLR ColsonC and VanPA (1976) DNA restriction andmodification systems in Salmonella SQ a new system derivedby recombination between the SB system of Salmonellatyphimurium and the SP system of Salmonella potsdam J GenMicrobiol 95 166ndash172

92 Fuller-PaceFV BullasLR DeliusH and MurrayNE (1984)Genetic recombination can generate altered restriction specificityProc Natl Acad Sci USA 81 6095ndash6099

93 NagarajaV ShepherdJC and BickleTA (1985) A hybridrecognition sequence in a recombinant restriction enzyme andthe evolution of DNA sequence specificity Nature 316 371ndash372

94 Fuller-PaceFV and MurrayNE (1986) Two DNA recognitiondomains of the specificity polypeptides of a family of type Irestriction enzymes Proc Natl Acad Sci USA 83 9368ndash9372

95 SchoulerC GautierM EhrlichSD and ChopinMC (1998)Combinational variation of restriction modification specificitiesin Lactococcus lactis Mol Microbiol 28 169ndash178

96 DybvigK SitaramanR and FrenchCT (1998) A family ofphase-variable restriction enzymes with differing specificitiesgenerated by high-frequency gene rearrangements Proc NatlAcad Sci USA 95 13923ndash13928

97 Cerdeno-TarragaAM PatrickS CrossmanLC BlakelyGAbrattV LennardN PoxtonI DuerdenB HarrisBQuailMA et al (2005) Extensive DNA inversions in the B fragilisgenome control variable gene expression Science 307 1463ndash1465

98 SzybalskiW KimSC HasanN and PodhajskaAJ (1991)Class-IIS restriction enzymesmdasha review Gene 100 13ndash26

99 BujnickiJM (2004) Molecular Phylogenetics of RestrictionEnzymes In PingoudA (ed) Restriction Enzymes Vol 14Springer Berlin New York pp 63ndash93

100 KongH and SmithCL (1997) Substrate DNA and cofactorregulate the activities of a multi-functional restriction-modification enzyme BcgI Nucleic Acids Res 25 3687ndash3692

101 SmithRM MarshallJJ JacklinAJ RetterSE HalfordSEand SobottF (2013) Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiesterbonds in DNA Nucleic Acids Res 41 391ndash404

102 SmithRM JacklinAJ MarshallJJ SobottF andHalfordSE (2013) Organization of the BcgI restriction-modification protein for the transfer of one methyl group toDNA Nucleic Acids Res 41 405ndash417

103 LacksS and GreenbergB (1975) A deoxyribonuclease ofDiplococcus pneumoniae specific for methylated DNA J BiolChem 250 4060ndash4066

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nloaded from

104 FriedhoffP LurzR LuderG and PingoudA (2001) Sau3AIa monomeric type II restriction endonuclease that dimerizes onthe DNA and thereby induces DNA loops J Biol Chem 27623581ndash23588

105 GreenePJ GuptaM BoyerHW BrownWE andRosenbergJM (1981) Sequence analysis of the DNA encodingthe Eco RI endonuclease and methylase J Biol Chem 2562143ndash2153

106 NewmanAK RubinRA KimSH and ModrichP (1981)DNA sequences of structural genes for Eco RI DNA restrictionand modification enzymes J Biol Chem 256 2131ndash2139

107 BougueleretL SchwarzsteinM TsugitaA and ZabeauM(1984) Characterization of the genes coding for the Eco RVrestriction and modification system of Escherichia coli NucleicAcids Res 12 3659ndash3676

108 TaoT and BlumenthalRM (1992) Sequence andcharacterization of pvuIIR the PvuII endonuclease gene and ofpvuIIC its regulatory gene J Bacteriol 174 3395ndash3398

109 BrooksJE NathanPD LandryD SznyterLA Waite-ReesP IvesCL MoranLS SlatkoBE and BennerJS(1991) Characterization of the cloned BamHI restrictionmodification system its nucleotide sequence properties of themethylase and expression in heterologous hosts Nucleic AcidsRes 19 841ndash850

110 McClarinJA FrederickCA WangBC GreenePBoyerHW GrableJ and RosenbergJM (1986) Structure ofthe DNA-Eco RI endonuclease recognition complex at 3 Aresolution Science 234 1526ndash1541

111 KimYC GrableJC LoveR GreenePJ and RosenbergJM(1990) Refinement of Eco RI endonuclease crystal structure arevised protein chain tracing Science 249 1307ndash1309

112 WinklerFK BannerDW OefnerC TsernoglouDBrownRS HeathmanSP BryanRK MartinPDPetratosK and WilsonKS (1993) The crystal structure ofEcoRV endonuclease and of its complexes with cognate andnon-cognate DNA fragments EMBO J 12 1781ndash1795

113 ChengX BalendiranK SchildkrautI and AndersonJE(1994) Structure of PvuII endonuclease with cognate DNAEMBO J 13 3927ndash3935

114 NewmanM StrzeleckaT DornerLF SchildkrautI andAggarwalAK (1994) Structure of restriction endonucleaseBamHI and its relationship to EcoRI Nature 368 660ndash664

115 AndersonJE (1993) Restriction endonucleases and modificationmethylases Curr Opin Struct Biol 3 24ndash30

116 AggarwalAK (1995) Structure and function of restrictionendonucleases Curr Opin Struct Biol 5 11ndash19

117 KovallRA and MatthewsBW (1998) Structural functionaland evolutionary relationships between lambda-exonuclease andthe type II restriction endonucleases Proc Natl Acad Sci USA95 7893ndash7897

118 HickmanAB LiY MathewSV MayEW CraigNL andDydaF (2000) Unexpected structural diversity in DNArecombination the restriction endonuclease connection MolCell 5 1025ndash1034

119 RobertsRJ (1976) Restriction endonucleases CRC Crit RevBiochem 4 123ndash164

120 RobertsRJ (1985) Restriction and modification enzymes andtheir recognition sequences Nucleic Acids Res 13(Suppl)r165ndashr200

121 RobertsRJ and MacelisD (1993) REBASEmdashrestrictionenzymes and methylases Nucleic Acids Res 21 3125ndash3137

122 AravindL MakarovaKS and KooninEV (2000) SURVEYAND SUMMARY holliday junction resolvases and relatednucleases identification of new families phyletic distribution andevolutionary trajectories Nucleic Acids Res 28 3417ndash3432

123 OrlowskiJ and BujnickiJM (2008) Structural and evolutionaryclassification of Type II restriction enzymes based on theoreticaland experimental analyses Nucleic Acids Res 36 3552ndash3569

124 BujnickiJM and RychlewskiL (2001) Grouping togetherhighly diverged PD-(DE)XK nucleases and identification ofnovel superfamily members using structure-guided alignment ofsequence profiles J Mol Microbiol Biotechnol 3 69ndash72

125 SokolowskaM CzapinskaH and BochtlerM (2011)Hpy188I-DNA pre- and post-cleavage complexesmdashsnapshots of

the GIY-YIG nuclease mediated catalysis Nucleic Acids Res39 1554ndash1564

126 CymermanIA ObarskaA SkowronekKJ LubysA andBujnickiJM (2006) Identification of a new subfamily of HNHnucleases and experimental characterization of a representativemember HphI restriction endonuclease Proteins 65 867ndash876

127 ChanSH OpitzL HigginsL OrsquoloaneD and XuSY (2010)Cofactor requirement of HpyAV restriction endonuclease PLoSOne 5 e9071

128 MakAN LambertAR and StoddardBL (2010) FoldingDNA recognition and function of GIY-YIG endonucleasescrystal structures of REco29kI Structure 18 1321ndash1331

129 SasnauskasG ConnollyBA HalfordSE and SiksnysV(2007) Site-specific DNA transesterification catalyzed by arestriction enzyme Proc Natl Acad Sci USA 104 2115ndash2120

130 MurrayIA StickelSK and RobertsRJ (2010) Sequence-specific cleavage of RNA by Type II restriction enzymes NucleicAcids Res 38 8257ndash8268

131 MochizukiA YaharaK KobayashiI and IwasaY (2006)Genetic addiction selfish genersquos strategy for symbiosis in thegenome Genetics 172 1309ndash1323

132 HadiSM BachiB IidaS and BickleTA (1983) DNArestrictionmdashmodification enzymes of phage P1 and plasmidp15B Subunit functions and structural homologies J MolBiol 165 19ndash34

133 RedaschiN and BickleTA (1996) Posttranscriptionalregulation of EcoP1I and EcoP15I restriction activityJ Mol Biol 257 790ndash803

134 KrugerDH KupperD MeiselA ReuterM and SchroederC(1995) The significance of distance and orientation of restrictionendonuclease recognition sites in viral DNA genomes FEMSMicrobiol Rev 17 177ndash184

135 MeiselA BickleTA KrugerDH and SchroederC (1992)Type III restriction enzymes need two inversely orientedrecognition sites for DNA cleavage Nature 355 467ndash469

136 HumbelinM SuriB RaoDN HornbyDP EberleHPripflT KenelS and BickleTA (1988) Type III DNArestriction and modification systems EcoP1 and EcoP15Nucleotide sequence of the EcoP1 operon the EcoP15 mod geneand some EcoP1 mod mutants J Mol Biol 200 23ndash29

137 BachiB ReiserJ and PirrottaV (1979) Methylation andcleavage sequences of the EcoP1 restriction-modification enzymeJ Mol Biol 128 143ndash163

138 PiekarowiczA and BrzezinskiR (1980) Cleavage andmethylation of DNA by the restriction endonuclease HinfIIIisolated from Haemophilus influenzae Rf J Mol Biol 144415ndash429

139 KrugerDH SchroederC ReuterM BogdarinaIGBuryanovYI and BickleTA (1985) DNA methylation ofbacterial viruses T3 and T7 by different DNA methylases inEscherichia coli K12 cells Eur J Biochem 150 323ndash330

140 MeiselA MackeldanzP BickleTA KrugerDH andSchroederC (1995) Type III restriction endonucleasestranslocate DNA in a reaction driven by recognition site-specificATP hydrolysis EMBO J 14 2958ndash2966

141 DrydenDT EdwardsonJM and HendersonRM (2011) DNAtranslocation by type III restriction enzymes a comparison ofcurrent models of their operation derived from ensemble andsingle-molecule measurements Nucleic Acids Res 39 4525ndash4531

142 CramptonN RoesS DrydenDT RaoDN EdwardsonJMand HendersonRM (2007) DNA looping and translocationprovide an optimal cleavage mechanism for the type IIIrestriction enzymes EMBO J 26 3815ndash3825

143 CramptonN YokokawaM DrydenDT EdwardsonJMRaoDN TakeyasuK YoshimuraSH and HendersonRM(2007) Fast-scan atomic force microscopy reveals that the typeIII restriction enzyme EcoP15I is capable of DNA translocationand looping Proc Natl Acad Sci USA 104 12755ndash12760

144 RamanathanSP vanAK SearsA PeakmanLJDiffinFM SzczelkunMD and SeidelR (2009) Type IIIrestriction enzymes communicate in 1D without looping betweentheir target sites Proc Natl Acad Sci USA 106 1748ndash1753

145 SchwarzFW vanAK TothJ SeidelR and SzczelkunMD(2011) DNA cleavage site selection by Type III restriction

Nucleic Acids Research 2014 Vol 42 No 1 15

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nloaded from

enzymes provides evidence for head-on protein collisionsfollowing 1D bidirectional motion Nucleic Acids Res 398024ndash8051

146 SzczelkunMD (2011) Translocation switching and gatingpotential roles for ATP in long-range communication on DNAby Type III restriction endonucleases Biochem Soc Trans 39589ndash594

147 GuptaYK YangL ChanSH SamuelsonJC XuSY andAggarwalAK (2012) Structural insights into the assembly andshape of Type III restriction-modification (R-M) EcoP15Icomplex by small-angle X-ray scattering J Mol Biol 420261ndash268

148 WyszomirskiKH CurthU AlvesJ MackeldanzP Moncke-BuchnerE SchutkowskiM KrugerDH and ReuterM (2012)Type III restriction endonuclease EcoP15I is a heterotrimericcomplex containing one Res subunit with several DNA-bindingregions and ATPase activity Nucleic Acids Res 40 3610ndash3622

149 RevelHR and uriaSE (1970) DNA-glucosylation in T-evenphage genetic determination and role in phagehost interactionAnnu Rev Genet 4 177ndash192

150 RevelHR (1983) DNA modification glucosylationIn MathewsCK KutterEM MosigG and BergetP (eds)Bacteriophage T4 American Society of MicrobiologyWashington DC pp 156ndash165

151 HattmanS and FukasawaT (1963) Host-induced modificationof T-even phages due to defective glucosylation of their DNAProc Natl Acad Sci USA 50 297ndash300

152 ShedlovskyA and BrennerS (1963) A chemical basis for thehost-induced modification of T-even bacteriophages Proc NatlAcad Sci USA 50 300ndash305

153 RevelHR HattmanS and LuriaSE (1965) Mutants ofbacteriophages T2 and T6 defective in alpha-glucosyl transferaseBiochem Biophys Res Commun 18 545ndash550

154 GeorgopoulosCP (1967) Isolation and preliminarycharacterization of T4 mutants with nonglucosylated DNABiochem Biophys Res Commun 28 179ndash184

155 GeorgopoulosCP and RevelHR (1971) Studies with glucosyltransferase mutants of the T-even bacteriophages Virology 44271ndash285

156 RevelHR (1967) Restriction of nonglucosylated T-evenbacteriophage properties of permissive mutants of Escherichiacoli B and K12 Virology 31 688ndash701

157 Noyer-WeidnerM DiazR and ReinersL (1986) Cytosine-specific DNA modification interferes with plasmid establishmentin Escherichia coli K12 involvement of rglB Mol Gen Genet205 469ndash475

158 RaleighEA and WilsonG (1986) Escherichia coli K-12 restrictsDNA containing 5-methylcytosine Proc Natl Acad Sci USA83 9070ndash9074

159 BlumenthalRM (1987) The PvuII restriction-modificationsystem cloning characterization and use in revealing an E colibarrier to certain methylases or methylated DNAIn ChirikjianJG (ed) Gene Amplification and Analysis Vol 5Elsevier New York pp 227ndash245

160 RaviRS SozhamannanS and DharmalingamK (1985)Transposon mutagenesis and genetic mapping of the rglA andrglB loci of Escherichia coli Mol Gen Genet 198 390ndash392

161 RaleighEA TrimarchiR and RevelH (1989) Genetic andphysical mapping of the mcrA (rglA) and mcrB (rglB) loci ofEscherichia coli K-12 Genetics 122 279ndash296

162 HeitmanJ and ModelP (1987) Site-specific methylases inducethe SOS DNA repair response in Escherichia coli J Bacteriol169 3243ndash3250

163 JanosiL YonemitsuH HongH and KajiA (1994) Molecularcloning and expression of a novel hydroxymethylcytosine-specificrestriction enzyme (PvuRts1I) modulated by glucosylation ofDNA J Mol Biol 242 45ndash61

164 BairCL and BlackLW (2007) A type IV modificationdependent restriction nuclease that targets glucosylatedhydroxymethyl cytosine modified DNAs J Mol Biol 366768ndash778

165 ZhouX HeX LiangJ LiA XuT KieserT HelmannJDand DengZ (2005) A novel DNA modification by sulphurMol Microbiol 57 1428ndash1438

166 LiuG OuHY WangT LiL TanH ZhouXRajakumarK DengZ and HeX (2010) Cleavage ofphosphorothioated DNA and methylated DNA by the type IVrestriction endonuclease ScoMcrA PLoS Genet 6 e1001253

167 RaleighEA MurrayNE RevelH BlumenthalRMWestawayD ReithAD RigbyPW ElhaiJ and HanahanD(1988) McrA and McrB restriction phenotypes of some E colistrains and implications for gene cloning Nucleic Acids Res 161563ndash1575

168 WoodcockDM CrowtherPJ DohertyJ JeffersonSDeCruzE Noyer-WeidnerM SmithSS MichaelMZ andGrahamMW (1989) Quantitative evaluation of Escherichia colihost strains for tolerance to cytosine methylation in plasmid andphage recombinants Nucleic Acids Res 17 3469ndash3478

169 GrahamMW DohertyJP and WoodcockDM (1990)Efficient construction of plant genomic libraries requires the useof mcr- host strains and packaging mixes Plant Mol Biol Rep8 33ndash42

170 BlumenthalRM GregorySA and CooperiderJS (1985)Cloning of a restriction-modification system from Proteusvulgaris and its use in analyzing a methylase-sensitive phenotypein Escherichia coli J Bacteriol 164 501ndash509

171 PalmerLE RabinowiczPD OrsquoShaughnessyAL BalijaVSNascimentoLU DikeS de la BastideM MartienssenRAand McCombieWR (2003) Maize genome sequencing bymethylation filtration Science 302 2115ndash2117

172 PennNW SuwalskiR OrsquoRileyC BojanowskiK and YuraR(1972) The presence of 5-hydroxymethylcytosine in animaldeoxyribonucleic acid Biochem J 126 781ndash790

173 PennNW (1976) Modification of brain deoxyribonucleic acidbase content with maturation in normal and malnourished ratsBiochem J 155 709ndash712

174 KriaucionisS and HeintzN (2009) The nuclear DNA base5-hydroxymethylcytosine is present in Purkinje neurons and thebrain Science 324 929ndash930

175 TahilianiM KohKP ShenY PastorWA BandukwalaHBrudnoY AgarwalS IyerLM LiuDR AravindL et al(2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosinein mammalian DNA by MLL partner TET1 Science 324930ndash935

176 OrdwayJM BedellJA CitekRW NunbergA GarridoAKendallR StevensJR CaoD DoergeRW KorshunovaYet al (2006) Comprehensive DNA methylation profiling in ahuman cancer genome identifies novel epigenetic targetsCarcinogenesis 27 2409ndash2423

177 IrizarryRA Ladd-AcostaC CarvalhoB WuHBrandenburgSA JeddelohJA WenB and FeinbergAP(2008) Comprehensive high-throughput arrays for relativemethylation (CHARM) Genome Res 18 780ndash790

178 LoenenWA DanielAS BraymerHD and MurrayNE(1987) Organization and sequence of the hsd genes ofEscherichia coli K-12 J Mol Biol 198 159ndash170

179 SzomolanyiE KissA and VenetianerP (1980) Cloning themodification methylase gene of Bacillus sphaericus R inEscherichia coli Gene 10 219ndash225

180 LiuY IchigeA and KobayashiI (2007) Regulation of theEcoRI restriction-modification system Identification of ecoRIMgene promoters and their upstream negative regulators in theecoRIR gene Gene 400 140ndash149

181 LiuY and KobayashiI (2007) Negative regulation of theEcoRI restriction enzyme gene is associated with intragenicreverse promoters J Bacteriol 189 6928ndash6935

182 MrukI LiuY GeL and KobayashiI (2011) Antisense RNAassociated with biological regulation of a restriction-modificationsystem Nucleic Acids Res 39 5622ndash5632

183 WardDF and MurrayNE (1979) Convergent transcription inbacteriophage lambda interference with gene expression J MolBiol 133 249ndash266

184 HeitmanJ ZinderND and ModelP (1989) Repair of theEscherichia coli chromosome after in vivo scission by the EcoRIendonuclease Proc Natl Acad Sci USA 86 2281ndash2285

185 TaoT BourneJC and BlumenthalRM (1991) A family ofregulatory genes associated with type II restriction-modificationsystems J Bacteriol 173 1367ndash1375

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186 IvesCL NathanPD and BrooksJE (1992) Regulation of theBamHI restriction-modification system by a small intergenicopen reading frame bamHIC in both Escherichia coli andBacillus subtilis J Bacteriol 174 7194ndash7201

187 SohailA IvesCL and BrooksJE (1995) Purification andcharacterization of CBamHI a regulator of the BamHIrestriction-modification system Gene 157 227ndash228

188 BartA DankertJ and van der EndeA (1999) Operatorsequences for the regulatory proteins of restriction modificationsystems Mol Microbiol 31 1277ndash1278

189 AntonBP HeiterDF BennerJS HessEJ GreenoughLMoranLS SlatkoBE and BrooksJE (1997) Cloning andcharacterization of the BgII restriction-modification systemreveals a possible evolutionary footprint Gene 187 19ndash27

190 RimselieneR VaisvilaR and JanulaitisA (1995) The eco72ICgene specifies a trans-acting factor which influences expression ofboth DNA methyltransferase and endonuclease from the Eco72Irestriction-modification system Gene 157 217ndash219

191 ZheleznayaLA KainovDE YunusovaAK andMatvienkoNI (2003) Regulatory C protein of the EcoRVmodification-restriction system Biochemistry (Mosc) 68105ndash110

192 CesnavicieneE MitkaiteG StankeviciusK JanulaitisA andLubysA (2003) Esp1396I restriction-modification systemstructural organization and mode of regulation Nucleic AcidsRes 31 743ndash749

193 HeidmannS SeifertW KesslerC and DomdeyH (1989)Cloning characterization and heterologous expression of theSmaI restriction-modification system Nucleic Acids Res 179783ndash9796

194 SemenovaE MinakhinL BogdanovaE NagornykhMVasilovA HeydukT SoloninA ZakharovaM andSeverinovK (2005) Transcription regulation of the EcoRVrestriction-modification system Nucleic Acids Res 336942ndash6951

195 McGeehanJE StreeterSD PapapanagiotouI FoxGC andKnealeGG (2005) High-resolution crystal structure of therestriction-modification controller protein CAhdI fromAeromonas hydrophila J Mol Biol 346 689ndash701

196 SawayaMR ZhuZ MershaF ChanSH DaburR XuSYand BalendiranGK (2005) Crystal structure of the restriction-modification system control element CBcll and mapping of itsbinding site Structure 13 1837ndash1847

197 BogdanovaE DjordjevicM PapapanagiotouI HeydukTKnealeG and SeverinovK (2008) Transcription regulation ofthe type II restriction-modification system AhdI Nucleic AcidsRes 36 1429ndash1442

198 CallowP SukhodubA TaylorJE and KnealeGG (2007)Shape and subunit organisation of the DNA methyltransferaseMAhdI by small-angle neutron scattering J Mol Biol 369177ndash185

199 MarksP McGeehanJ WilsonG ErringtonN and KnealeG(2003) Purification and characterisation of a novel DNAmethyltransferase MAhdI Nucleic Acids Res 31 2803ndash2810

200 MarksP McGeehanJ and KnealeG (2004) A novel strategyfor the expression and purification of the DNAmethyltransferase MAhdI Protein Expr Purif 37 236ndash242

201 McGeehanJE PapapanagiotouI StreeterSD andKnealeGG (2006) Cooperative binding of the CAhdIcontroller protein to the CR promoter and its role inendonuclease gene expression J Mol Biol 358 523ndash531

202 McGeehanJE StreeterS CooperJB MohammedFFoxGC and KnealeGG (2004) Crystallization andpreliminary X-ray analysis of the controller protein CAhdI fromAeromonas hydrophilia Acta Crystallogr D Biol Crystallogr60 323ndash325

203 PapapanagiotouI StreeterSD CaryPD and KnealeGG(2007) DNA structural deformations in the interaction of thecontroller protein CAhdI with its operator sequence NucleicAcids Res 35 2643ndash2650

204 StreeterSD PapapanagiotouI McGeehanJE andKnealeGG (2004) DNA footprinting and biophysicalcharacterization of the controller protein CAhdI

suggests the basis of a genetic switch Nucleic Acids Res 326445ndash6453

205 BallN StreeterSD KnealeGG and McGeehanJE (2009)Structure of the restriction-modification controller proteinCEsp1396I Acta Crystallogr D Biol Crystallogr 65 900ndash905

206 McGeehanJE StreeterSD ThreshSJ BallN RavelliRBand KnealeGG (2008) Structural analysis of the genetic switchthat regulates the expression of restriction-modification genesNucleic Acids Res 36 4778ndash4787

207 BallNJ McGeehanJE StreeterSD ThreshSJ andKnealeGG (2012) The structural basis of differential DNAsequence recognition by restriction-modification controllerproteins Nucleic Acids Res 40 10532ndash10542

208 McGeehanJE BallNJ StreeterSD ThreshSJ andKnealeGG (2012) Recognition of dual symmetry by thecontroller protein CEsp1396I based on the structure of thetranscriptional activation complex Nucleic Acids Res 404158ndash4167

209 BrooksJE BennerJS HeiterDF SilberKR SznyterLAJager-QuintonT MoranLS SlatkoBE WilsonGG andNwankwoDO (1989) Cloning the BamHI restrictionmodification system Nucleic Acids Res 17 979ndash997

210 MoranCP Jr LangN LeGriceSF LeeG StephensMSonensheinAL PeroJ and LosickR (1982) Nucleotidesequences that signal the initiation of transcription andtranslation in Bacillus subtilis Mol Gen Genet 186 339ndash346

211 NakayamaY and KobayashiI (1998) Restriction-modificationgene complexes as selfish gene entities roles of a regulatorysystem in their establishment maintenance and apoptoticmutual exclusion Proc Natl Acad Sci USA 95 6442ndash6447

212 SegalDJ and MecklerJF (2013) Genome engineering at thedawn of the golden age Annu Rev Genomics Hum Genet 14135ndash158

213 RambachA and TiollaisP (1974) Bacteriophage lambda havingEcoRI endonuclease sites only in the nonessential region of thegenome Proc Natl Acad Sci USA 71 3927ndash3930

214 ThomasM CameronJR and DavisRW (1974) Viablemolecular hybrids of bacteriophage lambda and eukaryoticDNA Proc Natl Acad Sci USA 71 4579ndash4583

215 KelleyWS ChalmersK and MurrayNE (1977) Isolation andcharacterization of a lambdapolA transducing phage Proc NatlAcad Sci USA 74 5632ndash5636

216 MurrayNE and KelleyWS (1979) Characterization oflambdapolA transducing phages effective expression of theE coli polA gene Mol Gen Genet 175 77ndash87

217 WilsonGG and MurrayNE (1979) Molecular cloning of theDNA ligase gene from bacteriophage T4 I Characterisation ofthe recombinants J Mol Biol 132 471ndash491

218 MurrayNE BruceSA and MurrayK (1979) Molecularcloning of the DNA ligase gene from bacteriophage T4 IIAmplification and preparation of the gene product J MolBiol 132 493ndash505

219 MidgleyCA and MurrayNE (1985) T4 polynucleotide kinasecloning of the gene (pseT) and amplification of its productEMBO J 4 2695ndash2703

220 SutcliffeJG (1978) pBR322 restriction map derived from theDNA sequence accurate DNA size markers up to 4361nucleotide pairs long Nucleic Acids Res 5 2721ndash2728

221 SutcliffeJG (1979) Complete nucleotide sequence of theEscherichia coli plasmid pBR322 Cold Spring Harb SympQuant Biol 43(Pt1) 77ndash90

222 KleinB and MurrayK (1979) Phage lambda receptorchromosomes for DNA fragments made with restrictionendonuclease I of Bacillus amyloliquefaciens H J Mol Biol133 289ndash294

223 LoenenWA and BrammarWJ (1980) A bacteriophage lambdavector for cloning large DNA fragments made with severalrestriction enzymes Gene 10 249ndash259

224 KarnJ BrennerS BarnettL and CesareniG (1980) Novelbacteriophage lambda cloning vector Proc Natl Acad SciUSA 77 5172ndash5176

225 ManiatisT HardisonRC LacyE LauerJ OrsquoConnellCQuonD SimGK and EfstratiadisA (1978) The isolation of

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structural genes from libraries of eucaryotic DNA Cell 15687ndash701

226 ManiatisT FritschA and SambrookJ (1982) MolecularCloning A Laboratory Manual Cold Spring Harbor LaboratoryCold Spring Harbor NY

227 HendrixRW RobertsJW StahlFW and WeisbergRA(eds) (1983) Lambda II Cold Spring Harbor Laboratory ColdSpring Harbor NY

228 StahlFW CrasemannJM and StahlMM (1975)Rec-mediated recombinational hot spot activity inbacteriophage lambda III Chi mutations are site-mutationsstimulating rec-mediated recombination J Mol Biol 94203ndash212

229 MoirA and BrammarWJ (1976) The use of specialisedtransducing phages in the amplification of enzyme productionMol Gen Genet 149 87ndash99

230 HohnB and MurrayK (1977) Packaging recombinant DNAmolecules into bacteriophage particles in vitro Proc Natl AcadSci USA 74 3259ndash3263

231 SternbergN TiemeierD and EnquistL (1977) In vitropackaging of a lambda Dam vector containing EcoRIDNA fragments of Escherichia coli and phage P1 Gene 1255ndash280

232 CollinsJ and HohnB (1978) Cosmids a type of plasmidgene-cloning vector that is packageable in vitro inbacteriophage lambda heads Proc Natl Acad Sci USA 754242ndash4246

233 Muller-HillB and KaniaJ (1974) Lac repressor can be fused tobeta-galactosidase Nature 249 561ndash563

234 KalninsA OttoK RutherU and Muller-HillB (1983)Sequence of the lacZ gene of Escherichia coli EMBO J 2593ndash597

235 MessingJ CreaR and SeeburgPH (1981) A system forshotgun DNA sequencing Nucleic Acids Res 9 309ndash321

236 MessingJ and VieiraJ (1982) A new pair of M13 vectors forselecting either DNA strand of double-digest restrictionfragments Gene 19 269ndash276

237 Yanisch-PerronC VieiraJ and MessingJ (1985) ImprovedM13 phage cloning vectors and host strains nucleotidesequences of the M13mp18 and pUC19 vectors Gene 33103ndash119

238 RobertsTM KacichR and PtashneM (1979) A generalmethod for maximizing the expression of a cloned gene ProcNatl Acad Sci USA 76 760ndash764

239 GuarenteL RobertsTM and PtashneM (1980) A techniquefor expressing eukaryotic genes in bacteria Science 2091428ndash1430

240 GuarenteL LauerG RobertsTM and PtashneM (1980)Improved methods for maximizing expression of a cloned genea bacterium that synthesizes rabbit beta-globin Cell 20543ndash553

241 OrsquoFarrellP PoliskyB and GelfandDH (1978) Regulatedexpression by readthrough translation from a plasmid-encodedbeta-galactosidase J Bacteriol 134 645ndash654

242 CasadabanMJ ChouJ and CohenSN (1980) In vitrogene fusions that join an enzymatically active beta-galactosidasesegment to amino-terminal fragments of exogenousproteins Escherichia coli plasmid vectors for the detectionand cloning of translational initiation signals J Bacteriol 143971ndash980

243 FriedL LassakJ and JungK (2012) A comprehensive toolboxfor the rapid construction of lacZ fusion reporters J MicrobiolMethods 91 537ndash543

244 LinnT and RallingG (1985) A versatile multiple- and single-copy vector system for the in vitro construction oftranscriptional fusions to lacZ Plasmid 14 134ndash142

245 LinnT and StPR (1990) Improved vector system forconstructing transcriptional fusions that ensures independenttranslation of lacZ J Bacteriol 172 1077ndash1084

246 PourcelC MarchalC LouiseA FritschA and TiollaisP(1979) Bacteriophage lambda-E coli K12 vector-host system forgene cloning and expression under lactose promoter control IDNA fragment insertion at the lacZ EcoRI restriction site MolGen Genet 170 161ndash169

247 UhlichGA and ChenCY (2012) A cloning vector for creationof Escherichia coli lacZ translational fusions and generation oflinear template for chromosomal integration Plasmid 67259ndash263

248 WindleBE (1986) Phage lambda and plasmid expressionvectors with multiple cloning sites and lacZ alpha-complementation Gene 45 95ndash99

249 YuD and CourtDL (1998) A new system to place singlecopies of genes sites and lacZ fusions on the Escherichia colichromosome Gene 223 77ndash81

250 HuynhTV YoungRA and DavisRW (1985) Constructingand screening cDNA libraries in lambda gt10 and lambda gt11In GloverDM (ed) DNA Cloning Vol 1 IRL Press OxfordUK pp 49ndash78

251 RutherU KoenenM SippelAE and Muller-HillB (1982)Exon cloning immunoenzymatic identification of exons of thechicken lysozyme gene Proc Natl Acad Sci USA 796852ndash6855

252 CupplesCG and MillerJH (1988) Effects of amino acidsubstitutions at the active site in Escherichia coli beta-galactosidase Genetics 120 637ndash644

253 CupplesCG and MillerJH (1989) A set of lacZ mutations inEscherichia coli that allow rapid detection of each of the sixbase substitutions Proc Natl Acad Sci USA 86 5345ndash5349

254 CupplesCG CabreraM CruzC and MillerJH (1990) A setof lacZ mutations in Escherichia coli that allow rapid detectionof specific frameshift mutations Genetics 125 275ndash280

255 GossenJA de LeeuwWJ and VijgJ (1994) LacZ transgenicmouse models their application in genetic toxicology MutatRes 307 451ndash459

256 ShwedPS CrosthwaitJ DouglasGR and SeligyVL (2010)Characterisation of MutaMouse lambdagt10-lacZ transgeneevidence for in vivo rearrangements Mutagenesis 25 609ndash616

257 GrodzickerT WilliamsJ SharpP and SambrookJ (1975)Physical mapping of temperature-sensitive mutations ofadenoviruses Cold Spring Harb Symp Quant Biol 39(Pt 1)439ndash446

258 HutchisonCA III NewboldJE PotterSS and EdgellMH(1974) Maternal inheritance of mammalian mitochondrial DNANature 251 536ndash538

259 PotterSS NewboldJE HutchisonCA III and EdgellMH(1975) Specific cleavage analysis of mammalian mitochondrialDNA Proc Natl Acad Sci USA 72 4496ndash4500

260 HellerR and ArnheimN (1980) Structure and organization ofthe highly repeated and interspersed 13 kb EcoRI-Bg1IIsequence family in mice Nucleic Acids Res 8 5031ndash5042

261 JeffreysAJ (1979) DNA sequence variants in the G gamma-A gamma- delta- and beta-globin genes of man Cell 18 1ndash10

262 JeffreysAJ CraigIW and FranckeU (1979) Localisationof the G gamma- A gamma- delta- and beta-globin geneson the short arm of human chromosome 11 Nature 281606ndash608

263 KanYW and DozyAM (1978) Polymorphism of DNAsequence adjacent to human beta-globin structural generelationship to sickle mutation Proc Natl Acad Sci USA 755631ndash5635

264 KanYW and DozyAM (1978) Antenatal diagnosis of sickle-cell anaemia by DNA analysis of amniotic-fluid cells Lancet2 910ndash912

265 BotsteinD WhiteRL SkolnickM and DavisRW (1980)Construction of a genetic linkage map in man using restrictionfragment length polymorphisms Am J Hum Genet 32314ndash331

266 GusellaJF WexlerNS ConneallyPM NaylorSLAndersonMA TanziRE WatkinsPC OttinaKWallaceMR SakaguchiAY et al (1983) A polymorphicDNA marker genetically linked to Huntingtonrsquos disease Nature306 234ndash238

267 WexlerNS (2012) Huntingtonrsquos disease advocacy drivingscience Annu Rev Med 63 1ndash22

268 GillP JeffreysAJ and WerrettDJ (1985) Forensic applicationof DNA lsquofingerprintsrsquo Nature 318 577ndash579

269 LindsayJA (2010) Genomic variation and evolution ofStaphylococcus aureus Int J Med Microbiol 300 98ndash103

18 Nucleic Acids Research 2014 Vol 42 No 1

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271 ArberW (1977) What is the function of restriction enzymesTrends Biochem Sci 2 N176ndashN178

272 IshikawaK HandaN and KobayashiI (2009) Cleavage of amodel DNA replication fork by a Type I restrictionendonuclease Nucleic Acids Res 37 3531ndash3544

273 IshikawaK HandaN SearsL RaleighEA and KobayashiI(2011) Cleavage of a model DNA replication fork by a methyl-specific endonuclease Nucleic Acids Res 39 5489ndash5498

274 McNeilEM and MeltonDW (2012) DNA repair endonucleaseERCC1-XPF as a novel therapeutic target to overcomechemoresistance in cancer therapy Nucleic Acids Res 409990ndash10004

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