We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.
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.