SfiI endonuclease activity is strongly influenced by the non-specificsequence in the middle of its recognition site

摘要

The Sfil endonuclease cleaves DNA at the sequence GGCCNNNN down arrow NGGCC, where N is any base and down arrow is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to Sfil, a series of BRA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which Sfil cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. Sfil cleaved some of these substrates at faster rates than other substrates, For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for k(cat) and K-m were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. Sfil activity is thus highly dependent on conformational variations in the spacer DNA.