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A number of structural and kinetic studies of DNA polymerases have proposed the catalytic mechanism of the nucleotidyl-transfer reaction. However, the actual process has never been visualized. Here we show the nucleotidyl-transfer reaction process catalyzed by human DNA polymerase η using time-resolved protein crystallography. In sequence, the nucleophile 3 ́-OH is deprotonated, the deoxyribose at the primer end converts from C2 ́-endo to C3 ́-endo, and the nucleophile and the α -phosphate of dATP approach each other to form the new bond. A third Mg2+ ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism. [ABSTRACT FROM AUTHOR]