Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Abstract

Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.hybrid ͉ AloI ͉ PpiI ͉ TstI R estriction endonucleases (REases) are parts of restrictionmodification (R-M) systems, whose primary biological function is the protection of bacterial cells from incoming foreign DNA molecules (1). There are three main groups of restriction enzymes (types I, II, and III), which differ in enzyme composition, cofactor requirements, and mode of action (2). The beststudied are type II REases, which in general recognize specific DNA targets of 4-8 bp and cleave DNA at or close to these sequences (1, 2). The exquisite accuracy of type II enzymes (3, 4) has made them indispensable tools for DNA manipulations. Although almost 3,700 type II REases with 262 different specificities have been characterized to date (5), there still is a demand for enzymes recognizing new DNA targets.During the past two decades, numerous efforts have been undertaken to engineer type II REases with altered specificities. Both rational protein design and random mutagenesis, followed by various selection procedures, have been tried (6), and several mutant enzymes with some preference for cleavage of altered DNA targets were isolated (7-10). However, projects concerned with orthodox type II REases so far have been largely unsuccessful mainly for two reasons: (i) difficulty of dealing with the observed tight coupling between DNA recognition and cleavage and (ii) absence of an efficient system for selecting enzymes with changed specificities. In this regard, unorthodox type II enzymes, such as the type IIG REase Eco57I (11), have shown more promise. Type IIG enzymes combine the catalytic centers of endonuclease and methyltransferase in one polypeptide chain, and the ability of Eco57I to methylate recognized DNA targets has been applied to isolate mutants having previously undescribed specificity (12).The discovery of AloI-like REases (13-15), classified as type IIB enzymes, opened up new opportunities for the engineering of type II REases with altered specificities. AloI-like REases ar...

Evolution of sequence specificity in a restriction endonuclease by a point mutation

“…Initially, the TRD swapping has been successfully applied to alter the DNA sequence specificity of Type I REases (5,7). Combinatorial reassortment of TRDs between Type IIB REases has been used more recently to generate a REase with novel DNA specificity (9). Yet another approach to obtain new specificity was to construct hybrid proteins that contain the DNA-binding and/or catalytic modules from different sources.…”

“…Later the reassortment of TRDs has been used to generate Type I REase with different specificity in the laboratory (5). Generation of new sequence specificity by TRD swapping was demonstrated in a Type II REase recently (9). Point mutations were also considered to contribute in evolving sequence specificity, but no examples have been documented so far (1,10).…”

Restriction Enzymes in Microbiology, Biotechnology and Biochemistry

“…By 1980, 56different specificities had been found (Roberts, 1980); and by 1990, over160 (Roberts, 1990). Today this number is around300, but the count matters less, now, because the specificities of certain Type II enzymes can be changed by domain swapping (Jurenaite-Urbanaviciene et al, 2007) and by rational mutagenesis (Morgan & Luyten, 2009), allowing potentially hundreds of new specificities to be created at will in the laboratory.…”

Bifunctional Prokaryotic DNA-Methyltransferases

“…This is perhaps why it is the only found example of natural bifunctional RMS originating from type II RMS. The newly found potential type IIC proteins are good candidates to expand on the current list of 12 bifunctional enzymes: AloI [10], BcgI [17], BseMII [18], BseRI [19], BspLU11III [20], CjeI [11], Eco57I [15], HaeIV [21], MmeI [12], PpiI [13], TstI [13] and TspGWI [14]. Taking into consideration intensiveness with what new microbial genomes have been sequencing during the last decade, new bifunctional RMS could be discovered very soon.…”

“…As can be seen from Table 1, the enzymes were grouped according to their domain organisation as it was presented in Conserved Domain Database [9]. As could be judged from their domain organisation, 20 new bifunctional REases are thought to represent the fusion of a REase with MTase and target recognition subunits of the type I restriction-modification systems (R-M-S structure), having similar organisation with the known type IIC bifunctional enzymes such as AloI [10], CjeI [11], MmeI [12], PpiI [13], TstI [13] and TspGWI [14]. Type I RMS enzymes are multisubunit proteins that function as a single protein complex, consisting of R, M and S subunits [3].…”