Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.Subtype IIS enzymes are a growing group of atypical REases that recognize a specific DNA sequence and cleave outside it at a defined distance, up to 21 nt, within any sequence [1,2]. Since their discovery, they have attracted considerable attention as objects of basic research in the field of protein-DNA interactions and as advanced tools for genetic engineering. One of the most intensively studied REases is FokI, specific to 5'-GGATG(N9/13)-3' sites, where asymmetry of the recognition site apparently imposes an unusual type of interaction with DNA: the large protein, monomeric in solution, transiently forms dimers and binds two recognition sites while the DNA loop is being generated [3]. Another subtype IIS REase - MmeI - not only cleaves DNA at 20/18 nt - one of the sites furthest removed from the recognition site - but also represents a model of a minimal restriction-modification system, where only one (the top) strand of the recognition site is methylated [4]. Molecular applications of subtype IIS enzymes,