The nucleotidyl transfer was initiated by transferring hPol η crystals to a pH 6.8 or 7.0 reaction buffer containing 1 mM Mg2+
but no dATP (Methods). After incubation at 293 K for 40s to 300s, the reaction was terminated in ~40s intervals by freezing crystals in liquid nitrogen at 77 K. Diffraction data were collected to 1.50 – 1.95 Å Bragg spacings (Supplementary Table 1
). The reaction process is monitored by the electron density corresponding to the new chemical bond in the Fo-Fc map compared with the refined GS structure (Supplementary Fig. 2
, ). The reaction time courses at pH 6.8 and 7.0 in crystallo
() are ~20–100 fold slower than in solution (), probably due to the reduced thermal motion. By 40s, the A-site was fully occupied with Mg2+
(, Supplementary Fig. 1c,d
). About 50% of Ca2+
in the B site was replaced by Mg2+
, and exchange of the remaining Ca2+
took place slowly (Supplementary Fig. 3
). Binding of two Mg2+
ions leads to the alignment of the 3′-OH and dATP 18,32
. The refined structure is nearly identical to that with dAMPNPP (PDB: 3MR2) except for tighter dATP coordination by R61 and closer of the Mg2+
ions (3.4 Å apart versus 3.6 Å). Since there is no sign of bond formation, the structure is termed the reactant state (RS).
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 (more ...)
Electron density corresponding to a new bond between the 3′-OH and the α-phosphorus of dATP begins to emerge at 80 seconds, increases quickly in the following 60s, and reaches its maximum after 200s when the reaction is 60–70% complete (). The seemingly reduced rate after 140s is likely due to the reverse reaction in crystallo
. The slight decline of product after 250s () is due to a sideway product translocation, which is clear with longer incubation (Supplementary Fig 4a
). To alleviate an impediment to proper DNA translocation by the crystal lattice, we replaced an AT basepair with a mismatch at the DNA end that formed lattice contacts (Methods, Supplementary Table 2
). One DNA with a TG mismatch led to isomorphous crystals and proper translocation of the DNA product (Supplementary Fig 4b
). Interestingly, the time courses of nucleotidyl transfer in the AT and TG crystals are nearly identical and unaffected by the lattice contacts ().
At the peak of chemical bond formation between 200 and 250s, the scissile phosphate can be refined in a penta-covalent transition state without restraints (). But the bond distances between the phosphorus and the attacking or leaving oxygen atoms are 2.2 to 2.5 Å (), much longer than the expected 2.0 Å observed with transition-state mimics like AlF4
. Moreover, in the Fo-Fc map residual electron densities are observed around the new and scissile phosphodiester bonds (). The same diffraction data, however, can be well fitted as a mixture of the reactant (RS) and the product state (PS) immediately before and after the nucleotidyl transfer (). Because the transition state is transient and unstable, the structures obtained between 80s and 300s are refined as a mixture of the RS and PS at different ratios (, Supplementary Table 1a
). For instance the 1.52 Å structure at 230s contains 40% substrate and 60% product complexes.
Between the RS and PS, the protein, DNA and dATP are superimposable except for atoms in the reaction center. Most notably, the α-phosphorus of the dATP moves 1.4 Å along a straight line between the attacking and leaving oxygen atoms (separated by 4.6 Å) (). The shift of the α-phosphate is accompanied by alteration of R61 of hPolη, which flips away from the scissile phosphate and is replaced by a new metal ion and water molecules (, see details below). On the primer strand, changes are confined to the 3′ nucleotide. The 3′-OH together with the deoxyribose moves towards the α-phosphate by 0.5 Å, and the sugar pucker changes from C2′ endo in the RS to C3′ endo in the PS (). With the loss of the nucleophile and α-phosphate as ligands, the A-site Mg2+
dissociates in the PS as evidenced by the declining occupancy (Supplementary Fig 1c,d
). Concomitantly, D13 assumes a second conformation and forms an H-bond with K224.
The C3′-endo (A-form) conformation at the 3′ primer end was observed in ternary complexes with the A, B and X-family DNA polymerases and thought to be important for forming a shallow minor-groove for dNTP selection 9,11,12,14,15,17,18,32
. Among the Y-family polymerases, the primer end has always been observed as C2′-endo28
. In the hPol η GS and RS complexes, the dATP and the nucleotide 5′ to the primer end have the A-form conformation, but only in the PS structure does the primer end adopt the C3′-endo conformation to avoid clashes between its C2′ atom and the non-bridging oxygen of dATP during and immediately after the nucleophilic attack (, ). Since the electron density is weak for the sugar moiety at the 3′-primer end, we tested the effect of the A-form conformation by using a primer with a ribonucleotide at its 3′ end. The catalytic efficiency (kcat
) of hPol ηis comparable whether it is rA- or dA (), as observed for DNA pol β36
, indicating that the A-form conformation is probably necessary for DNA synthesis in general.
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 (more ...)