Scheme 1.

The nucleotide incorporation pathway catalyzed by the T4 DNA polymerase. Complexes II and III are pre-insertion complexes that are formed very rapidly in the presence of the correct nucleotide. If 2AP is the templating base, complex II is fluorescent, but 2AP fluorescence is quenched in complex III, which indicates conformational changes between complexes II and III. The pre-insertion complexes are likely open complexes that facilitate rapid nucleotide binding. Further conformational changes are required to produce complex IV, a closed complex, which is formed before the chemistry step of phosphodiester bond formation.