Watching DNA polymerase η make a phosphodiester bond - PubMed (original) (raw)

Watching DNA polymerase η make a phosphodiester bond

Teruya Nakamura et al. Nature. 2012.

Abstract

DNA synthesis has been extensively studied, but the chemical reaction itself has not been visualized. Here we follow the course of phosphodiester bond formation using time-resolved X-ray crystallography. Native human DNA polymerase η, DNA and dATP were co-crystallized at pH 6.0 without Mg(2+). The polymerization reaction was initiated by exposing crystals to 1 mM Mg(2+) at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg(2+) ions are aligned within 40 s, but the bond formation is not evident until 80 s. From 80 to 300 s structures show a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicate that deprotonation and an accompanying C2'-endo to C3'-endo conversion of the nucleophile 3'-OH are rate limiting. A third Mg(2+) 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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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig 1

Fig 1

pH and metal ion dependence of hPol η. (a) Diagrams of DNA synthesis and the procedure of the in crystallo reaction. AH and B stands for general acid and base, respectively. (b) Reaction rate (kobs) is plotted against mixed Mg2+ and Ca2+ at the total of 1mM. (c) The pH and buffer dependent reaction rates. (d) The GS structure. The active site is superimposed with the 2Fo-Fc electron density contoured at 1.2σ (upper panel) or with the non-reactive dAMPNPP ternary complex (PDB: 3MR2, silver, lower panel). Hydrogen bonds and metal ion coordination are shown as dashed lines.

Fig. 2

Fig. 2

Reaction time course. (a) Two views (upper and lower panels) of omit Fo40-230s - Fc40s maps (4.0σ) superimposed onto the 40s structure (pH 7.0). The emerging densities are pointed out or circled. (b) A plot of the absolute peak height of the new bond density versus reaction time at pH 6.8 and 7.0 in AT and TG crystals (Supplementary Table 1a,b). The noise level (1σ) is indicated. (c) The 230s structure refined as the pentacovalent transition state (TS), or (d) the RS (yellow) and PS (blue) mixture. The 2Fo-Fc (1σ, grey) and Fo-Fc map (±3.0σ green and red) are superimposed.

Fig. 3

Fig. 3

Deprotonation of the 3′-OH. (a) Superposition of the refined RS (80s, yellow) and PS (300s, blue). The transient water molecule, likely deprotonating the 3′-OH, is circled. The sugar pucker at the primer 3′-end changes from C2′-endo in the RS to C3′-endo in the PS. The clashes between the α-phosphate and the C2′ if it is C2′-endo, and between the C2′ and the transient water if the water doesn’t depart, are indicated by dashed double arrowheads. (b) WT and S113A mutant hPol η can both extend primer with a ribonucleotide at the 3′end (lighter color) as with pure DNA (darker color).

Fig. 4

Fig. 4

The 3rd Mg2+ ion in hPol η catalysis. A stereo view of the three metal ions observed in the intermediate state of 230s after Mg2+ addition, which consists of a mixture of PS (blue) and RS (yellow). The 3rd Mg2+ has six ligands, four of which are water molecules. The 2Fo-Fc map contoured at 1.5 σ (grey) and Fo-Fc map with the 3rd Mg2+ omitted contoured at 4.0 σ (green) are overlaid with the ball-and-stick model.

Fig. 5

Fig. 5

A proposed mechanism for metal-ion dependent polymerase reaction. The B-site metal ion is stably associated with the incoming dNTP and the enzyme. Binding of Mg2+ at the A-site aligns the reactants, in particular the 3′-OH, and promotes its deprotonation, which is assisted by the dNTP and the transient water. After the reaction is initiated and before the products are released, a third metal ion replaces R61 to stabilize the reaction intermediates.

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