Engineering of Restriction Endonucleases: Using Methylation Activity of the Bifunctional Endonuclease Eco57I to Select the Mutant with a Novel Sequence Specificity

Rimšeliene, Renata; Manelienė, Z.; Lubys, Arvydas; Janulaitis, A.

doi:10.1016/s0022-2836(03)00142-6

Cited by 32 publications

(28 citation statements)

References 31 publications

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“…For example, isolated catalytic domains of Type IIS REases (R.FokI and R.BmrI) were fused with specific DNA-binding domains of transcriptional regulators, to generate chimeric endonucleases with altered specificities (15)(16)(17). A selection system (methylation activity-based selection) has also been developed to produce REases with new specificity (18). This method appears to be of limited application because it can only be used on bifunctional type IIG REases (19).…”

Section: Resultsmentioning

confidence: 99%

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

Matheshwaran

Vasu

Nagaraja

2008

Proc. Natl. Acad. Sci. U.S.A.

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Restriction endonucleases (REases) protect bacteria from invading foreign DNAs and are endowed with exquisite sequence specificity. REases have originated from the ancestral proteins and evolved new sequence specificities by genetic recombination, gene duplication, replication slippage, and transpositional events. They are also speculated to have evolved from nonspecific endonucleases, attaining a high degree of sequence specificity through point mutations. We describe here an example of generation of exquisitely site-specific REase from a highly-promiscuous one by a single point mutation.genetic recombination ͉ protein engineering ͉ R.Kpnl ͉ metal ion coordination ͉ promiscuous activity T ype II REases are part of restriction-modification (R-M) systems that protect bacterial cells against invading foreign genomes (1). The enzymes are highly sequence specific and cleave DNA at the target sites several orders of magnitude more readily than at noncanonical sequences. Because of their high sequence specificity they play crucial role in DNA manipulation and characterization. Of the 3,700 Type II REases identified from different bacterial species so far, only 262 specificities have been characterized (2). The growth in the applications of REases in DNA manipulations warranted engineering of these enzymes possessing novel specificities, and numerous efforts have been undertaken toward this goal. These attempts to engineer REases with altered specificities were largely unsuccessful because of the high degree of sequence specificity already attained through specific contacts to the bases by different amino acid residues from various structural segments of the enzyme. This would imply that changing the specificity may require modification of several of the contacts with the bases and the phosphate backbone.In nature, evolution of sequence specificity in R-M systems is achieved through genetic recombination, gene duplication, replication slippage, and transposition (3-8). Genetic recombination occurring in nature to reassort target recognition domains (TRDs) leading to evolution of sequence specificity was demonstrated in Salmonella Type I R-M systems (3, 4). 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).In this study, we provide evidence for point mutations having a role in evolving sequence specificity in a REase. Although R.KpnI is a typical Type IIP REase, it exhibits prolific promiscuous cleavage. In the presence of Mg 2ϩ , the most common metal ion required for REases, R.KpnI cleaves plasmid DNA at variety of noncanonical sites in a robust fashion, generating extensive DNA fragmentation. We describe here a single point mutant, which carries out DNA cleavage in an extremely sitespecific manner, confirming t...

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Section: Resultsmentioning

confidence: 99%

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

Matheshwaran

Vasu

Nagaraja

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…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).…”

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confidence: 99%

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

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…D22, probably originated from the fusion of type III enzymes. It has conserved motifs similar to Eco57I protein, which consists of Mod and Res subunits of type III enzymes [15]. Type III systems are composed of two genes (mod and res) encoding protein subunits that function in one protein complex either in DNA recognition and modification, Mod, or restriction, Res [3].…”

Section: Methyltransferase Fusions With a Restriction Endonucleasementioning

confidence: 99%

“…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.…”

Section: Methyltransferase Fusions With a Restriction Endonucleasementioning

confidence: 99%

Bifunctional Prokaryotic DNA-Methyltransferases

Nikitin¹,

Kertész‐Farkas²,

Solonin³

et al. 2012

Methylation - From DNA, RNA and Histones to Diseases and Treatment

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Engineering of Restriction Endonucleases: Using Methylation Activity of the Bifunctional Endonuclease Eco57I to Select the Mutant with a Novel Sequence Specificity

Cited by 32 publications

References 31 publications

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

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

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

Bifunctional Prokaryotic DNA-Methyltransferases

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