CMLS, Cell. Mol. Life Sci. 62 (2005) 685–707 1420-682X/05/060685-23 DOI 10.1007/s00018-004-4513-1 CMLS Cellular and Molecular Life Sciences © Birkhäuser Verlag, Basel, 2005 Review Type II restriction endonucleases: structure and mechanism A. Pingouda,*, M. Fuxreiterb, V.Pingouda and W.Wendea a Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, 35392 Giessen (Germany), Fax: +49 641 9935409, e-mail: [email protected] b Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, Karolina ut 29, 1113 Budapest (Hungary) Received 15 November 2004; accepted 9 December 2004 Abstract. Type II restriction endonucleases are compo- few restriction endonucleases discovered thus far do not nents of restriction modification systems that protect belong to the PD…D/ExK family of enzymes, but rather bacteria and archaea against invading foreign DNA. Most have active sites typical of other endonuclease families. are homodimeric or tetrameric enzymes that cleave DNA The present review deals with new developments in the at defined sites of 4–8 bp in length and require Mg2+ ions field of Type II restriction endonucleases. One of the for catalysis. They differ in the details of the recognition more interesting aspects is the increasing awareness of process and the mode of cleavage, indicators that these the diversity of Type II restriction enzymes. Nevertheless, enzymes are more diverse than originally thought. Still, structural studies summarized herein deal with the more most of them have a similar structural core and seem to common subtypes. A major emphasis of this review will share a common mechanism of DNA cleavage, suggest- be on target site location and the mechanism of catalysis, ing that they evolved from a common ancestor. Only a two problems currently being addressed in the literature. Key words. Protein-nucleic acid interaction; facilitated diffusion; DNA recognition; DNA cleavage; mechanism of phosphodiester bond hydrolysis; evolution; protein engineering. Introduction among bacteria [4] and generation of genetic variation [5, 6]. Restriction endonucleases of Chlorella viruses may Restriction endonucleases are components of restriction have a nutritive function by helping degrade host DNA or modification (RM) systems that occur ubiquitously preventing infection of a cell by another virus [3]. Certain among bacteria, archaea [1, 2] and in viruses of certain types of RM systems can also be considered as selfish unicellular algae [3]. Their main function is to defend DNA elements [7, 8]. In general, bacteria and archaea their host against foreign DNA. This is achieved by cleav- harbour numerous RM systems. For example, in Heli- ing incoming DNA that is recognized as foreign by cobacter pylori more than 20 putative RM systems, the absence of a characteristic modification (N4 or C5 comprising greater than 4% of the total genome, havebeen methylation at cytosine or N6 methylation at adenine) at identified in two completely sequenced H. pylori strains defined sites within the recognition sequence. The host [9]. Several types of restriction endonucleases exist that DNA is resistant to cleavage as these sites are modified. differ in subunit composition and cofactor requirement. Additional functions have been attributed to restriction Commonly, four types are distinguished [10]. enzymes, including maintenance of species identity Type I restriction enzymes consist of three different subunits, HsdM, HsdR and HsdS, that are responsible for * Corresponding author. modification, restriction and sequence recognition, 686 A. Pingoud et al. Type II restriction endonucleases respectively. The quaternary structure of the active Type I DNA within this sequence in both strands, producing restriction enzyme is HsdM2HsdR2HsdS. Type I enzymes 3¢-hydroxyls and 5¢-phosphate ends. Some recognize require ATP, Mg2+and AdoMet for activity. They interact discontinuous palindromes, interrupted by a segment of in general with two asymmetrical bi-partite recognition specified length but unspecified sequence. The DNA sites, translocate the DNA in an ATP-hydrolysis depen- fragments produced have ‘blunt’ or ‘sticky’ ends with dent manner and cut the DNA distal to the recognition 3¢- or 5¢-overhangs of up to 5 nucleotides (there is a sites, approximately half-way between two sites. Typical single known example of an enzyme producing a 7-nu- examples are EcoKI, EcoAI, EcoR124I and StySBLI, cleotide 3¢-overhang: TspRI (CASTGNN/)). Most of the which represent Type IA, IB, IC and ID subtypes, respec- restriction enzymes used for recombinant DNA work tively [11–14]. [21–23] belong to this subtype, which is called Type IIP Type III restriction enzymes consist of two subunits only, (P for –palindromic) according to the accepted nomencla- Mod (responsible for DNA recognition and modification) ture [10]. Many Type II restriction endonucleases have and Res (responsible for DNA cleavage). Active nucleases properties different from the Type IIP enzymes, for which 2+ have a Mod2Res2 stoichiometry, require ATP and Mg for EcoRI (recognition sequence G/AATTC) and EcoRV activity and are stimulated by AdoMet. They interact with (GAT/ATC) are the best-known and best-studied repre- two head-to-head arranged asymmetrical recognition sentatives. The current nomenclature tries to group the sites, translocate the DNA in an ATP-hydrolysis depen- Type II restriction enzymes according to properties that dent manner and cut the DNA close to one recognition are unique to the respective subtype. However, as will be site. Typical examples are EcoP1I and EcoP15I [12–14]. seen, overlap cannot be avoided. This is the consequence Type IV restriction enzymes recognize and cleave methy- of the great diversity among Type II restriction endonu- lated DNA. As such they are not part of an RM system. cleases. The best-studied representative is McrBC, which consists Type IIA enzymes recognize asymmetric sequences. An of two different subunits, McrB and McrC, responsible interesting member of this subtype is Bpu10I [CCT- for DNA recognition and cleavage, respectively. McrBC NAGC(-5/-2)], a dimer of non-identical subunits, each of recognizes DNA with at least two RC sequences at a vari- which is responsible for cleavage of one strand of the able distance, containing methylated or hydroxymethy- DNA: 5¢-CC/TNAGC-3¢ and 5¢-GC/TNAGG-3¢ [24]. lated cytosine in one or both strands. For DNA cleavage These enzymes are ideal precursors for the generation of GTP and Mg2+ are required; cleavage occurs close to one nicking enzymes. of the two RmC sites [12–14]. Type IIB enzymes cleave DNA at both sides of the This review will deal with Type II restriction endonu- recognition sequence, an example being BplI cleases with a particular focus on the structure and [(8/13)GAGNNNNNCTC(13/8)]. BplI cleaves the top mechanism of these enzymes. For previous reviews see strand 8 nucleotides before and 13 nucleotides after the references [15–20]. recognition sequence, while the bottom strand is cleaved 13 nucleotides before and 8 nucleotides after the recogni- tion sequence [25]. Diversity of Type II restriction endonucleases Type IIC enzymes have both cleavage and modification domains within one polypeptide. One of the first discov- As of 29 October 2004, REBASE (http://rebase.neb.com/ ered was BcgI [(10/12)CGANNNNNNTGC(12/10)], rebase/rebase.html) lists 3707 restriction enzymes: 59 which has a very unusual functional organization: it has Type I, 3635 Type II, 10 Type III and 3 Type IV. The an A2B quaternary structure [26] with both endonuclease predominance of Type II enzymes certainly is biased by and methyltransferase domains in the A subunit and the their usefulness for recombinant DNA work. The analysis target recognition domain located in the B subunit [27]. of published genome sequences suggests a somewhat BcgI illustrates the problem of the nomenclature of Type more even distribution among putative RM systems: II restriction endonucleases, as it is also a Type IIB approximately 29% Type I, 45% Type II, 8% Type III and enzyme. 18% Type IV [R. Roberts, personal comm.]. Type IIE enzymes need to interact with two copies of Type II restriction endonucleases differ from the Type I, their recognition sequence for efficient cleavage, one III and IV enzymes by a more simplified subunit organi- copy being the target for cleavage, the other serving as an zation. They are usually homodimeric or homotetrameric allosteric effector [28–30]. The best-studied examples enzymes that cleave DNA within or close to their recog- with respect to structure and function are EcoRII nition site and do not require ATP or GTP. With only one (/CCWGG) [31-36] and NaeI (GCC/CGG) [37-41]. It is exception known to date (see below), they require Mg2+ interesting to note that the removal of the effector domain as cofactor. of EcoRII converts this Type IIE enzyme into a very Orthodox Type II enzymes are homodimers that recognizes active Type IIP enzyme [34]. Sau3AI (/GATC), in the palindromic sequences of 4–8 bp in length, and cleave absence of DNA a monomer with two similar domains, CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Review Article 687 dimerizes in the presence of DNA and then functions as a which is composed of two different subunits. The func- Type IIE enzyme, with a catalytic site and an allosteric tional restriction endonuclease presumably is a a2b2 effector site [42]. tetramer [68]. Several of these enzymes have been used to Type IIF enzymes are typically homotetrameric restric- generate nicking enzymes, viz. BbvCI, BsaI, BsmAI, tion endonucleases that also interact with two copies of BsmBI and BsrDI [69]. their recognition site, but cleave both of them in a more Some Type II restriction enzymes only nick DNA. DNA or less concerted manner [28, 30, 43, 44]. Well-studied nicking endonucleases were found in Bacillus stearother- examples are Cfr10I (R/CCGGY) [45-47], NgoMIV mophilus, for example Nt.BstNBI and Nt.BstSEI (G/CCGGC) [47, 48] and SfiI (GGCCNNNN/NGGCC) (GAGTCN4/) [70, 71], and in Chlorella viruses, for exam- [49, 50].