Pyrophosphate release is a necessary step in nucleotide incorporation, allowing dNTPs to be used. These are the form of nucleotides present in the cell, and are stable under physiological conditions. On incorporation, two of the three phosphate groups must be cleaved off to allow the formation of the sugar-phosphate backbone. This results in the release of PPi.
The phosphate is only able to form 5 bonds – pentavalent – and if the two other phosphates remained attached to the nucleotide it would destabilise the structure. The two oxygens only bound to the phosphate have a slightly double bond character, increasing their stability. This also prevents them from being able to bond to other groups.
Mg2+ ions are necessary in arranging the DNA correctly, ensuring it is orientated in the correct manner to allow for efficient catalysis. The metal ions also stabilise the molecules, preventing the PPi molecule from causing damage before it degrades to 2 Pi. The formation of bonds with the metal ions in the polymerase are able to be observed in ‘real-time’ through X-ray crystallography time lapses, with different polymerases, at different stages of the reaction process. This allows the mechanism, and involvement of the ions, to be studied in detail.
In some cases, a third metal ion may be present. It does not have a well understood function at the moment.