Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography.

Structures of enzyme-substrate/product complexes have been studied for over four decades but have been limited to either before or after a chemical reaction. Recently using in crystallo catalysis combined with X-ray diffraction, we have discovered that many enzymatic reactions in nucleic acid metabolism require additional metal ion cofactors that are not present in the substrate or product state. By controlling metal ions essential for catalysis, the in crystallo approach has revealed unprecedented details of reaction intermediates. Here we present protocols used for successful studies of Mg2+-dependent DNA polymerases and ribonucleases that are applicable to analyses of a variety of metal ion-dependent reactions.