TstI, a Type II restriction-modification protein with DNA recognition, cleavage and methylation functions in a single polypeptide

Abstract

Type II restriction–modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conven-tional Type II schemes as they employ the same pro-tein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study anal-ysed one such single-chain protein, TstI. Compar-ison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the het-erologous assembly of BcgI, TstI forms a homote-tramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites