Characterization of AloI, a restriction-modification system of a new type

Cesnaviciene, E.; Petrušyté, M.; Kazlauskienė, Rita; Manelienė, Z.; Timinskas, Albertas; Lubys, Arvydas; Janulaitis, A.

doi:10.1006/jmbi.2001.5049

Cited by 33 publications

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“…Genes for PpiI and TstI were cloned and expressed in Escherichia coli [see Cloning of the Gene Encoding the PpiI RestrictionModification System and Cloning of the Gene Encoding the TstI Restriction-Modification System in supporting information (SI) Text], and the cloning and expression of the aloI gene were conducted as described (13). The primary structure analysis revealed very similar organization among AloI, PpiI, and TstI.…”

Section: Characterization Of Reases Used For Domain-swapping Experimementioning

confidence: 99%

“…each enzyme, indicating the potential involvement of this region in the DNA hydrolysis reaction (13). The central portions of the polypeptides are made up of conserved sequence motifs characteristic for amino methyltransferases (21) and presumably participate in AdoMet binding and the DNA methylation reaction.…”

Section: Characterization Of Reases Used For Domain-swapping Experimementioning

confidence: 99%

“…The discovery of AloI-like REases (13)(14)(15), classified as type IIB enzymes, opened up new opportunities for the engineering of type II REases with altered specificities. AloI-like REases are large polypeptides having both DNA endonuclease and methyltransferase activities.…”

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confidence: 99%

“…AloI-like REases are large polypeptides having both DNA endonuclease and methyltransferase activities. This group of enzymes recognize bipartite DNA targets and cleave DNA on both sides of recognition sequences (13)(14)(15). It turned out that C-terminal regions of AloI-like REases share sequence similarity with specificity (HsdS) subunits of some type I R-M systems.…”

mentioning

confidence: 99%

“…It turned out that C-terminal regions of AloI-like REases share sequence similarity with specificity (HsdS) subunits of some type I R-M systems. Based on this observation, it was predicted that C-terminal regions of AloI-like REases, just like HsdS subunits, have two TRDs, each recognizing an individual part of the bipartite DNA target (13).…”

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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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Section: Characterization Of Reases Used For Domain-swapping Experimementioning

confidence: 99%

Section: Characterization Of Reases Used For Domain-swapping Experimementioning

confidence: 99%

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