Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily - PubMed (original) (raw)

Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily

Mindaugas Zaremba et al. J Mol Biol. 2004.

Abstract

The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence. Unlike other restriction enzymes, it functions without metal ions. The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily. Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically. BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer. On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer. The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second. Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus. It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily. BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence.

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Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily - PubMed (original) (raw)