Raymond J. Williams - SGGW

1 MOLECULAR BIOTECHNOLOGYV olume 23, 2003 Restriction Endonucleases225225 Molecular Biotechnology 2003 Humana Press Inc. All rights of any nature whatsoever reserved. 1073 6085/2003/23:3/225 243/$ IntroductionRestriction endonucleases, which cleave bothstrands of DNA in a site-specific manner, are a fun-damental tool of molecular biology. Discovery ofendonucleases began in the 1960s and led to com-mercial availability in the early 1970s. The numberof characterized enzymes continues to grow as doesthe number of vendors and the size of their productlines. Although many similarities exist amongendonucleases in terms of structures, mechanisms,and uses, important differences remain. Now astaple of molecular biology, restriction endonu-cleases remain an area of active research regardingtheir cleavage mechanism, in vivo function, evolu-tionary origins, and as a model for site-specificDNA recognition.

2 New native enzymes continue tobe discovered, known enzymes cloned, and newendonuclease activities developed by using proteinengineering and fusions to produce novel Diversity and In Vivo FunctionAlthough primarily found in bacterial genomesand plasmids, restriction endonucleases also existin archaea, viruses, and eukaryotes. It is estimatedthat 1 in 4 bacteria examined contain one or more(1). Neisseria and Helicobacter pylori are particu-larly rich sources for multiple enzymes in a singlestrain. Respectively, as many as 7 and 14 endonu-clease genes have been discovered in individualstrains, although some of the genes are not activelyexpressed (2,3). Including all types, 3500 restric-tion enzymes that recognize 259 different DNAsequences are now known.

3 The vast majority ofthese, approx 3460 enzymes recognizing 234 DNAsequences, are classified as orthodox Type II orType II subclasses. These are the common tools ofmolecular biology with more than 500 enzymescomprising over 200 specificities commerciallyavailable. In addition, 58 homing endonucleases,so-called because they are encoded by genes thatAbstractRestriction endonucleases have become a fundamental tool of molecular biology with many commercialvendors and extensive product lines. While a significant amount has been learned about restriction enzymediversity, genomic organization, and mechanism, these continue to be active areas of research and assist inclassification efforts. More recently, one focus has been their exquisite specificity for the proper recognitionsequence and the lack of homology among enzymes recognizing the same DNA sequence.

4 Some questionsalso remain regarding in vivo function. Site-directed mutagenesis and fusion proteins based on known endo-nucleases show promise for custom-designed cleavage. An understanding of the enzymes and their proper-ties can improve their productive application by maintaining critical digest parameters and enhancing oravoiding alternative Entries: Restriction endonucleases; R/M systems; star activity; single-stranded cleavage; site-spe-cific EndonucleasesClassification, Properties, and ApplicationsRaymond J. WilliamsREVIEW*Author to whom all correspondence and reprint requests should be addressed: Protein Purification Dept., Promega Corp., 2800 WoodsHollow Road, Madison, WI 53711-5399. 9:54 AM225 MOLECULAR BIOTECHNOLOGYV olume 23, 2003226 Williamsare mobile, self-splicing introns or inteins, eachwith a unique recognition site, have been discov-ered.

5 A total of 16 site-specific nickases are cur-rently known as well. In all, 297 restrictionenzymes have been cloned and sequenced. A data-base of all known endonucleases is updatedmonthly by Dr. Richard J. Roberts and DanaMacelis and is available at A number of formats are available, refer-ences given, and statistics endonucleases were originally namedfor their ability to restrict the growth of phage in ahost bacterial cell by cleavage of the invading this manner, they may be acting as bacterial pro-tection systems. The DNA of the host is protectedfrom restriction by the activity of a methylase(s),which recognizes the same sequence as the restric-tion enzyme and methylates a specific nucleotide(4-methylcytosine, 5-methylcytosine, 5-hydroxy-methylcytosine, or 6-methyladenine) on each strandwithin this sequence.

6 Once methylated, the hostDNA is no longer a substrate for the both strands of the host DNA are methy-lated and even hemi-methylated DNA is protected,freshly replicated host DNA is not digested by role of restriction endonucleases as a pro-tection system may be oversimplified characteristics lower an enzyme s protec-tive potential. There would be no effect on phageswithout at least a dsDNA intermediate or those forwhom the DNA was also modified at the criticalbases. A small number of phage may be methy-lated by the host before restriction can occur, andthus be able to propagate protectively methylatedcopies of themselves. Also, large enzyme recog-nition sites would tend to be rare in small phagegenomes. Restriction site avoidance appears to bemore important in a group of bacteria rather thana corresponding group of phage.

7 The endonu-cleases generally have a longer half-life than thecorresponding methylases, a potentially lethalproblem for the host if the methylase is not prop-erly maintained. For these reasons, it has also beenproposed that restriction-methylase systems maybe mobile, selfish genetic elements that becomeessential for host survival once acquired (4,5). 2. Nomenclature and GenomicOrganizationIndividual enzymes are named in accordancewith the proposal of Smith and Nathans (6).Briefly, three letters in italics are derived from thefirst letter of the genus and the first two letters ofthe microbial species from which the enzyme wasderived. An additional letter without italics maybe used to designate a particular strain. This is fol-lowed by a roman numeral to signify the first, sec-ond, and so on, enzyme discovered from theorganism.

8 As may be deduced from the large num-ber of enzymes and the limited number of differ-ent DNA sequences they recognize, manyenzymes from different biological sources recog-nize the same DNA sequence and are calledisoschizomers. A subset wherein two enzymesrecognize the same DNA sequence but cleave at adifferent position is referred to as important point to emphasize as a result ofcloning and sequence comparison is that little ifany sequence homology exists between the endo-nuclease and methyltransferase recognizing thesame DNA sequence. Furthermore, even restric-tion isoschizomers may show little or no homol-ogy, including the amino acids involved inrecognition, and as such are excellent candidatesfor a comparative study of protein DNA interac-tion.

9 For example, the enzymes HhaII and HinfIare both isolated from strains of Haemophilus,recognize GANTC, and cleave between the G andA. However, they share only 19% identity in theiramino acid sequence (7). Endonuclease/methylasesystems recognizing the same sequence may alsoexhibit different methylation patterns and restric-tion sensitivity. Only a limited common aminoacid motif, , has been proven bymutational or structural analysis to participate incatalysis for 10 endonucleases. However, the 10enzymes include members that are classified asType II, IIe, IIs, IV, or intron encoded endonu-cleases (8). In contrast, general motifs have been04/JW549/ Williams /225-2442/10/03, 9:54 AM226 MOLECULAR BIOTECHNOLOGYV olume 23, 2003 Restriction Endonucleases227found for 30 6-methyladenine, 4-methylcytosine,and 5-methylcytosine methylases (9).

10 Frequently referred to as an R/M system, therestriction endonuclease and modification methy-lase genes lie adjacent to each other on bacterialDNA and may be oriented transcriptionally in aconvergent, divergent, or sequential manner. Theproximity of these genes appears to be universaland is utilized in a common cloning method some-times referred to as the Hungarian Trick (10).Basically, an endonuclease is used to digest thegenomic DNA from the bacteria containing the R/M system of interest and create a library of expression vector used must contain the rec-ognition site of the R/M system. Purified plasmidsfrom the clones are then subjected to the restric-tion enzyme of interest in vitro. If a plasmid con-tains the expressed methylase gene, it will beresistant to cleavage.