Abstract
Enzyme catalysis has been studied for over a century. How it actually occurs has not been visualized until recently. By combining in crystallo reaction and X-ray diffraction analysis of reaction intermediates, we have obtained unprecedented atomic details of the DNA synthesis process. Contrary to the established theory that enzyme-substrate complexes and transition states have identical atomic composition and catalysis occurs by the two-metal-ion mechanism, we have discovered that an additional divalent cation has to be captured en route to product formation. Unlike the canonical two metal ions, which are coordinated by DNA polymerases, this third metal ion is free of enzyme coordination. Its location between the α- and β-phosphates of dNTP suggests that the third metal ion may drive the phosphoryltransfer from the leaving group opposite to the 3′-OH nucleophile. Experimental data indicate that binding of the third metal ion may be the rate-limiting step in DNA synthesis and the free energy associated with the metal-ion binding can overcome the activation barrier to the DNA synthesis reaction.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0118-2) contains supplementary material, which is available to authorized users.
Highlights
When the R61A mutant Pol η was examined in crystallo, we found that binding of the A and B site metal ions occurred within 40 s as for WT, but without R61 the incoming dNTP is misaligned relative to the 3′-OH by 0.3 Å
Comparison of the existing DNA polymerases, reverse transcriptases and RNA polymerases demonstrates that the active site compositions are highly conserved. Because both DNA pol η and pol β, which differ in tertiary structures, require the conserved third metal ion in catalysis, we propose that the third metal ion is a general feature and all polymerization reactions of nucleic acids occur by three-metal-ion catalysis (Additional file 2: Movie S2)
The different environment surrounding the third metal ion, which is in a stark contrast to the conserved environment surrounding the canonical A and B site metal ions, gives hope that the third metal ion Extended from kinetic and structural studies, which lay the ground work for understanding DNA synthesis reactions, our in crystallo reaction and time resolved X-ray diffraction analysis has led to the discovery of the third metal ion and its capture being the rate-limiting step of DNA synthesis reaction
Summary
In crystallo catalysis of DNA synthesis Because DNA synthesis is pH and metal ion dependent, by reducing pH to 6.0 and using the non-catalytic metalion cofactor Ca2+, crystals of native DNA Pol η in complex with DNA substrate and correct incoming dNTP can be grown over a couple of weeks without reaction taking place [24]. A third metal ion is essential for DNA synthesis In both in crystallo catalysis by Pol η and β, a third divalent cation coordinated by reaction products is observed (Fig. 1b–d).
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