Abstract
Native human DNA polymerase η, DNA and dATP were crystallized at pH 6.0 without Mg2+. The polymerization reaction was initiated by exposing crystals to 1 mM Mg2+ at pH 7.0, and stopped at various time points by freezing at 77 K for crystallographic analysis. The substrates and two Mg2+ are aligned for reaction within 40 seconds, but the new bond begins to form at 80 seconds. Structures at reaction times from 80s to 300s reveal a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. In sequence, the nucleophile 3′-OH is deprotonated, the deoxyribose at the primer end converts from C2′-endo to C3′-endo to avoid steric clashes, 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.
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