Thomas D. Christian

Raman difference spectroscopy is used to monitor the conformational changes associated with dNMP incorporation in single crystals of a RB69 DNA polymerase (RB69pol) ternary complex. An inactive crystal of L415G RB69pol exo‐ with a primer/template duplex and a cognate dATP was produced in the presence of Ca²⁺. The Raman spectrum of the crystallized complex was obtained using a Raman microscope. Phosphodiester bond formation is triggered by soaking in Mg²⁺. The Raman difference spectra [ie, (ternary complex + Mg²⁺)–(ternary complex with no Mg²⁺)] show that conformational changes are complete prior to the acquisition of the first Raman spectrum following magnesium incorporation into the crystal. Polyacrylamide gel electrophoresis experiments carried in parallel show that dNMP incorporation occurs rapidly in the crystal. The Raman data indicate that significant changes in protein amide features, tyrosine ring modes, and nucleic acid base backbone modes have occurred. Overall, they are dominated by the dichroic effect with the molecular groups changing orientation with respect to the laser beam following nucleotide incorporation. The amide I and amide III α‐helix features are unusually narrow and are assigned to movement of the P and N helices. These results demonstrate the ability of Raman spectroscopy to monitor conformational changes in proteins via dichroism. The Raman spectra and the gel electrophoresis support the hypothesis that the major spectroscopic changes are because of structural changes in the ternary complex crystal following correct dNMP incorporation. Copyright © 2015 John Wiley & Sons, Ltd.

Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2′-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 102–103-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 102–103-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n – 2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.

The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3′-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Förster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

Benzo[a]pyrene (B[a]P) is a potent environmental carcinogen that is metabolized into diol epoxides that react with exocyclic amines in DNA. These DNA adducts have been shown to block DNA replication by high-fidelity polymerases and induce both base substitution and frame-shift mutations. To improve our understanding of the molecular mechanism of B[a]P-induced mutagenesis, a fluorescence resonance energy transfer (FRET) method was developed in which the (+)- or (−)-trans-anti-B[a]P−N²-dG adducts, positioned in the active site of DNA polymerase I (Klenow fragment), serve as donor fluorophores to an acceptor molecule positioned on the DNA primer strand. FRET was measured for a primer that ended one nucleotide before the adduct position and one that ended across from the adduct and used to estimate the distances between the two fluorophores. These estimates are consistent with prior studies that suggest the adducts are positioned in the minor groove. A comparison of the FRET for the (+)- and (−)-trans-B[a]P adducts in the Klenow active site suggested that the (+)-trans adduct is positioned ∼2 Å farther from the acceptor, consistent with the structural differences observed in duplex DNA where it has been shown that the (+)-trans adduct is oriented toward the 5′-end of the template strand while the (−)-trans adduct lies toward the 3′-end. Surprisingly, the adduct position did not change significantly when the primer was one nucleotide longer. The addition of either a correct (dCTP) or incorrect nucleotides showed only minor differences in FRET, suggesting that the adduct did not undergo a large change in the position within the polymerase active site, as expected if the adduct inhibited the polymerase conformational change.