Restriction Endonucleasesreview 225

Restriction EndonucleasesREVIEW 225 Restriction Endonucleases Classification, Properties, and Applications Raymond J. Williams Abstract Restriction endonucleases have become a fundamental tool of molecular biology with many commercial vendors and extensive product lines. While a significant amount has been learned about restriction enzyme diversity, genomic organization, and mechanism, these continue to be active areas of research and assist in classification efforts. More recently, one focus has been their exquisite specificity for the proper recognition sequence and the lack of homology among enzymes recognizing the same DNA sequence. Some questions also remain regarding in vivo function. Site-directed mutagenesis and fusion proteins based on known endonucleases show promise for custom-designed cleavage. An understanding of the enzymes and their properties can improve their productive application by maintaining critical digest parameters and enhancing or avoiding alternative activities. Index Entries: Restriction endonucleases; R/M systems; star activity; single-stranded cleavage; site-specific nickases. 1. Introduction 1.1. Diversity and In Vivo Function Restriction endonucleases, which cleave both Although primarily found in bacterial genomes strands of DNA in a site-specific manner, are a fun- and plasmids, restriction endonucleases also exist damental tool of molecular biology. Discovery of in archaea, viruses, and eukaryotes. It is estimated endonucleases began in the 1960s and led to com- that 1 in 4 bacteria examined contain one or more mercial availability in the early 1970s. The number (1). Neisseria and Helicobacter pylori are particu- of characterized enzymes continues to grow as does larly rich sources for multiple enzymes in a single the number of vendors and the size of their product strain. Respectively, as many as 7 and 14 endonu- lines. Although many similarities exist among clease genes have been discovered in individual endonucleases in terms of structures, mechanisms, strains, although some of the genes are not actively and uses, important differences remain. Now a expressed (2,3). Including all types, Ͼ3500 restric- staple of molecular biology, restriction endonu- tion enzymes that recognize 259 different DNA cleases remain an area of active research regarding sequences are now known. The vast majority of their cleavage mechanism, in vivo function, evolu- these, approx 3460 enzymes recognizing 234 DNA tionary origins, and as a model for site-specific sequences, are classified as orthodox Type II or DNA recognition. New native enzymes continue to Type II subclasses. These are the common tools of be discovered, known enzymes cloned, and new molecular biology with more than 500 enzymes endonuclease activities developed by using protein comprising over 200 specificities commercially engineering and fusions to produce novel poly- available. In addition, 58 homing endonucleases, peptides. so-called because they are encoded by genes that *Author to whom all correspondence and reprint requests should be addressed: Protein Purification Dept., Promega Corp., 2800 Woods Hollow Road, Madison, WI 53711-5399. E-mail:[email protected]. Molecular Biotechnology 2003 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2003/23:3/225–243/$20.00 MOLECULAR BIOTECHNOLOGY 225 Volume 23, 2003 04/JW549/Williams/225-244 225 2/10/03, 9:54 AM 226 Williams are mobile, self-splicing introns or inteins, each be mobile, selfish genetic elements that become with a unique recognition site, have been discov- essential for host survival once acquired (4,5). ered. A total of 16 site-specific nickases are cur- rently known as well. In all, 297 restriction 2. Nomenclature and Genomic enzymes have been cloned and sequenced. A data- Organization base of all known endonucleases is updated Individual enzymes are named in accordance monthly by Dr. Richard J. Roberts and Dana with the proposal of Smith and Nathans (6). Macelis and is available at http://www.neb.com/ Briefly, three letters in italics are derived from the rebase. A number of formats are available, refer- first letter of the genus and the first two letters of ences given, and statistics maintained. the microbial species from which the enzyme was Restriction endonucleases were originally named derived. An additional letter without italics may for their ability to restrict the growth of phage in a be used to designate a particular strain. This is fol- host bacterial cell by cleavage of the invading DNA. lowed by a roman numeral to signify the first, sec- In this manner, they may be acting as bacterial pro- ond, and so on, enzyme discovered from the tection systems. The DNA of the host is protected organism. As may be deduced from the large num- from restriction by the activity of a methylase(s), ber of enzymes and the limited number of differ- which recognizes the same sequence as the restric- ent DNA sequences they recognize, many tion enzyme and methylates a specific nucleotide enzymes from different biological sources recog- (4-methylcytosine, 5-methylcytosine, 5-hydroxy- nize the same DNA sequence and are called methylcytosine, or 6-methyladenine) on each strand isoschizomers. A subset wherein two enzymes within this sequence. Once methylated, the host recognize the same DNA sequence but cleave at a DNA is no longer a substrate for the endonuclease. different position is referred to as neoschizomers. Because both strands of the host DNA are methy- An important point to emphasize as a result of lated and even hemi-methylated DNA is protected, cloning and sequence comparison is that little if freshly replicated host DNA is not digested by the any sequence homology exists between the endo- endonuclease. nuclease and methyltransferase recognizing the The role of restriction endonucleases as a pro- same DNA sequence. Furthermore, even restric- tection system may be oversimplified however. tion isoschizomers may show little or no homol- Various characteristics lower an enzyme’s protec- ogy, including the amino acids involved in tive potential. There would be no effect on phages recognition, and as such are excellent candidates without at least a dsDNA intermediate or those for for a comparative study of protein–DNA interac- whom the DNA was also modified at the critical tion. For example, the enzymes HhaII and HinfI bases. A small number of phage may be methy- are both isolated from strains of Haemophilus, lated by the host before restriction can occur, and recognize GANTC, and cleave between the G and thus be able to propagate protectively methylated A. However, they share only 19% identity in their copies of themselves. Also, large enzyme recog- amino acid sequence (7). Endonuclease/methylase nition sites would tend to be rare in small phage systems recognizing the same sequence may also genomes. Restriction site avoidance appears to be exhibit different methylation patterns and restric- more important in a group of bacteria rather than tion sensitivity. Only a limited common amino a corresponding group of phage. The endonu- acid motif, PD...D/EXK, has been proven by cleases generally have a longer half-life than the mutational or structural analysis to participate in corresponding methylases, a potentially lethal catalysis for 10 endonucleases. However, the 10 problem for the host if the methylase is not prop- enzymes include members that are classified as erly maintained. For these reasons, it has also been Type II, IIe, IIs, IV, or intron encoded endonu- proposed that restriction-methylase systems may cleases (8). In contrast, general motifs have been MOLECULAR BIOTECHNOLOGY Volume 23, 2003 04/JW549/Williams/225-244 226 2/10/03, 9:54 AM Restriction Endonucleases 227 found for 30 6-methyladenine, 4-methylcytosine, as the PvuII C gene (12). Why some C genes stimu- and 5-methylcytosine methylases (9). late expression of alternate endonucleases is not Frequently referred to as an R/M system, the fully understood, but the phenomenon may have restriction endonuclease and modification methy- evolutionary implications for R/M systems. lase genes lie adjacent to each other on bacterial 3. Structure, Specificity, and Mechanism DNA and may be oriented transcriptionally in a convergent, divergent, or sequential manner. The 3.1. Classification and General Mechanism proximity of these genes appears to be universal Restriction endonucleases are classified and is utilized in a common cloning method some- according to their structure, recognition site, times referred to as the “Hungarian Trick” (10). cleavage site, cofactor(s), and activator(s). Sets of Basically, an endonuclease is used to digest the these criteria are used to define the different types genomic DNA from the bacteria containing the R/ (I, II, III, and IV) and subclasses (IIe, IIf, IIs, etc.), M system of interest and create a library of clones. which are explained in detail in Table 1. Multiple The expression vector used must contain the rec- subunit and holoenzyme assemblies are possible ognition site of the R/M system. Purified plasmids to achieve the needed restriction, methylase, and from the clones are then subjected to the restric- specificity domains. These three domains may be tion enzyme of interest in vitro. If a plasmid con- present on three separate polypeptides, two tains the expressed methylase gene, it will be polypeptides, or a single polypeptide. At a mini- resistant to cleavage. Often, the endonuclease is mum, all R/M systems share an absolute require- expressed as well without the need for subcloning. ment of Mg2ϩ for endonuclease activity and It is assumed that methylation must occur AdoMet (also referred to as S-adenosyl methion- before restriction activity to protect the host DNA. ine) as the methyl donor for methylase activity. In One approach bacteria use to limit the possibility general, Type I restriction requires Mg2ϩ, of self-restriction is to significantly reduce the AdoMet, and ATP (which becomes hydrolyzed). number of recognition sites in their genomes. Type II restriction requires only Mg2ϩ, although a Alternatively, methylase expression may precede second recognition site or AdoMet may be stimu- that of the endonuclease.

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