A new method for determining the stereochemistry of DNA cleavage reactions: Application to the SfiI and HpaII restriction endonucleases and to the MuA transposase

A new method was developed for tracking the stereochemical path of enzymati c cleavage of DNA, DNA with a phosphorothioate of known chirality at the sc issile bond is cleaved by the enzyme in (H2O)-O-18. The cleavage produces a DNA molecule with the 5'-[O-16,O-18,S]-thiophosphoryl group, whose chirali ty depends pn:whether the cleavage reaction proceeds by a single-step hydro lysis mechanism or by a-two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorot hioate dictates whether the O-18 is retained or lost in the ligation produc t, which can be determined by mass spectrometry. This method has advantages over previews methods in that it is not restricted to particular NA sequen ces, requires substantially less material, and avoids purification of the p roducts at intermediate stages in the procedure. The method was validated b y confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposas e. In all three cases, DNA cleavage proceeded with inversion of configurati on, indicating direct hydrolysis of the phosphodiester bond by water as opp osed to a reaction involving a covalent enzyme-DNA intermediate.