Bioactivity and mechanisms of flavonoids in decreasing insulin resistance.

Flavonoids are ubiquitous compounds in nature and are found in many Chinese herbal medicines. Due to their biological activity, flavonoids show potential for decreasing insulin resistance (IR), thereby delaying the progression of diabetes and accompanying metabolic syndromes. This review focuses on the mechanisms of flavonoids decreasing IR: (1) the interaction between flavonoids and target proteins of the insulin signalling pathway; (2) bioactivities of flavonoids, such as anti-inflammatory, lipid-lowering and antioxidant. Meanwhile, we summarise the structural characteristics, structure activity relationships and biological activity of flavonoids, providing evidence for their potential in the treatment of IR. Here, we also analyse the potential and limitations of their therapeutic use.

Insight into the Tumor Suppression Mechanism from the Structure of Human Polypyrimidine Splicing Factor (PSF/SFPQ) Complexed with a 30mer RNA from Murine Virus-like 30S Transcript-1.

Human polypyrimidine-binding splicing factor (PSF/SFPQ) is a tumor suppressor protein that regulates the gene expression of several proto-oncogenes and binds to the 5'-polyuridine negative-sense template (5'-PUN) of some RNA viruses. The activity of PSF is negatively regulated by long-noncoding RNAs, human metastasis associated in lung adenocarcinoma transcript-1 and murine virus-like 30S transcript-1 (VL30-1). PSF is a 707-amino acid protein that has a DNA-binding domain and two RNA recognition motifs (RRMs). Although the structure of the apo-truncated PSF is known, how PSF recognizes RNA remains elusive. Here, we report the 2.8 Å and 3.5 Å resolution crystal structures of a biologically active truncated construct of PSF (sPSF, consisting of residues 214-598) alone and in a complex with a 30mer fragment of VL30-1 RNA, respectively. The structure of the complex reveals how the 30mer RNA is recognized at two U-specific induced-fit binding pockets, located at the previously unrecognized domain-swapped, inter-subunit RRM1 (of the first subunit)-RRM2 (of the second subunit) interfaces that do not exist in the apo structure. Thus, the sPSF dimer appears to have two conformations in solution: one in a low-affinity state for RNA binding, as seen in the apo-structure, and the other in a high-affinity state for RNA binding, as seen in the sPSF-RNA complex. PSF undergoes an all or nothing transition between having two or no RNA-binding pockets. We predict that the RNA binds with a high degree of positive cooperativity. These structures provide an insight into a new regulatory mechanism that is likely involved in promoting malignancies and other human diseases.

Structural Insights into Binding of Remdesivir Triphosphate within the Replication-Transcription Complex of SARS-CoV-2.

Remdesivir is an adenosine analogue that has a cyano substitution in the C1' position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1' atom with its strong electron-withdrawing cyano group. Within the replication-transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.

Two-Metal-Ion Catalysis: Inhibition of DNA Polymerase Activity by a Third Divalent Metal Ion.

Almost all DNA polymerases (pols) exhibit bell-shaped activity curves as a function of both pH and Mg2+ concentration. The pol activity is reduced when the pH deviates from the optimal value. When the pH is too low the concentration of a deprotonated general base (namely, the attacking 3'-hydroxyl of the 3' terminal residue of the primer strand) is reduced exponentially. When the pH is too high the concentration of a protonated general acid (i.e., the leaving pyrophosphate group) is reduced. Similarly, the pol activity also decreases when the concentration of the divalent metal ions deviates from its optimal value: when it is too low, the binding of the two catalytic divalent metal ions required for the full activity is incomplete, and when it is too high a third divalent metal ion binds to pyrophosphate, keeping it in the replication complex longer and serving as a substrate for pyrophosphorylysis within the complex. Currently, there is a controversy about the role of the third metal ion which we will address in this review.

Insights into Binding of Single-Stranded Viral RNA Template to the Replication-Transcription Complex of SARS-CoV-2 for the Priming Reaction from Molecular Dynamics Simulations.

A minimal replication-transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5'-end to the last 3'-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3'-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5'-UUUAU-3' next to stem-loop (SL) 1'. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.

The effect of different divalent cations on the kinetics and fidelity of Bacillus stearothermophilus DNA polymerase.

Although Mg2+ is the metal ion that functions as the cofactor for DNA polymerases (DNA pols) in vivo, Mn2+ can also serve in this capacity but it reduces base discrimination. Metal ions aside from Mg2+ or Mn2+ can act as cofactors for some DNA pols but not for others. Here we report on the ability of several divalent metal ions to substitute for Mg2+ or Mn2+ with BST DNA polymerase (BST pol), an A family DNA pol. We selected the metal ions based on whether they had previously been shown to be effective with other DNA pols. We found that Co2+ and Cd2+ were the only cations tested that could replace Mg2+ or Mn2+. When Co2+ was substituted for Mg2+, the incorporation efficiency for correct dNTPs increased 6-fold but for incorrect dNTPs there was a decrease which depended on the incoming dNTP. With Mn2+, base selectivity was impaired compared to Co2+ and Cd2+. In addition, Co2+ and Mn2+ helped BST pol to catalyze primer-extension past a mismatch. Finally both Co2+ and Mn2+ enhanced ground-state binding of both correct and incorrect dNTPs to BST pol: Dideoxy terminated primer-template complexes.

Single-molecule FRET reveals proofreading complexes in the large fragment of Bacillus stearothermophilus DNA polymerase I.

There is increasing interest in the use of DNA polymerases (DNA pols) in next-generation sequencing strategies. These methodologies typically rely on members of the A and B family of DNA polymerases that are classified as high-fidelity DNA polymerases. These enzymes possess the ability to selectively incorporate the correct nucleotide opposite a templating base with an error frequency of only 1 in 106 insertion events. How they achieve this remarkable fidelity has been the subject of numerous investigations, yet the mechanism by which these enzymes achieve this level of accuracy remains elusive. Several smFRET assays were designed to monitor the conformational changes associated with the nucleotide selection mechanism(s) employed by DNA pols. smFRET has also been used to monitor the movement of DNA pols along a DNA substrate as well as to observe the formation of proof-reading complexes. One member among this class of enzymes, the large fragment of Bacillus stearothermophilus DNA polymerase I (Bst pol I LF), contains both 5'→3' polymerase and 3'→5' exonuclease domains, but reportedly lacks exonuclease activity. We have designed a smFRET assay showing that Bst pol I LF forms proofreading complexes. The formation of proofreading complexes at the single molecule level is strongly influenced by the presence of the 3' hydroxyl at the primer-terminus of the DNA substrate. Our assays also identify an additional state, observed in the presence of a mismatched primer-template terminus, that may be involved in the transfer of the primer-terminus from the polymerase to the exonuclease active site.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases.

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B, for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3'-hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group, making it a highly effective nucleophile that can attack the α phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly, two metal ions are required for proofreading; one helps to lower the pKa of the attacking water molecule, and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all, of the effects of these different metal ions can be rationalized based on their intrinsic properties, which are tabulated in this review.

Effect of Different Divalent Cations on the Kinetics and Fidelity of RB69 DNA Polymerase.

Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.

RB69 DNA polymerase structure, kinetics, and fidelity.

This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n - 1 and n - 2 of the primer strand and position n - 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°-tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.

Mispairs with Watson-Crick base-pair geometry observed in ternary complexes of an RB69 DNA polymerase variant.

Recent structures of DNA polymerase complexes with dGMPCPP/dT and dCTP/dA mispairs at the insertion site have shown that they adopt Watson-Crick geometry in the presence of Mn(2+) indicating that the tautomeric or ionization state of the base has changed. To see whether the tautomeric or ionization state of base-pair could be affected by its microenvironment, we determined 10 structures of an RB69 DNA polymerase quadruple mutant with dG/dT or dT/dG mispairs at position n-1 to n-5 of the Primer/Template duplex. Different shapes of the mispairs, including Watson-Crick geometry, have been observed, strongly suggesting that the local environment of base-pairs plays an important role in their tautomeric or ionization states.

Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics.

Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in other B family pols resulted in higher mutation rates. When similar substitutions for L415 were introduced into RB69pol, only L415A and L415G had dramatic effects on pre-steady-state kinetic parameters, reducing base selectivity by several hundred fold. On the other hand, the L415M variant behaved like the wild-type. Using a novel tC(o)-tCnitro Förster Resonance Energy Transfer (FRET) assay, we were able to show that the partition of the primer terminus between pol and exonuclease (exo) domains was compromised with the L415A and L415G mutants, but not with the L415M variant. These results could be rationalized by changes in their structures as determined by high resolution X-ray crystallography.

DNA mismatch synthesis complexes provide insights into base selectivity of a B family DNA polymerase.

Current hypotheses that attempt to rationalize the high degree of base selectivity exhibited by replicative DNA polymerases (pols) concur that ternary complexes formed with incorrect dNTPs are destabilized. Knowing what accounts for this destabilization is likely to be the key to understanding base discrimination. To address this issue, we have determined crystal structures of ternary complexes with all 12 mismatches using an engineered RB69 pol quadruple mutant (qm, L415A/L561A/S565G/Y567A) that enabled us to capture nascent mispaired dNTPs. These structures show that mismatches in the nascent base-pair binding pocket (NBP) of the qm pol differ markedly from mismatches embedded in binary pol-DNA complexes. Surprisingly, only 3 of 12 mismatches clash with the NBP when they are modeled into the wild-type (wt) pol. The remaining can fit into a wt pol ternary complex but deviate from normal Watson-Crick base-pairs. Repositioning of the templating nucleotide residue and the enlarged NBP in qm ternary complex play important roles in accommodating incorrect incoming dNTPs. From these structures, we propose additional reasons as to why incorrect dNTPs are incorporated so inefficiently by wt RB69 pol: (i) steric clashes with side chains in the NBP after Fingers closing; (ii) weak interactions or large gaps between the incoming dNTP and the templating base; and (iii) burying a protonated base in the hydrophobic environment of the NBP. All of these possibilities would be expected to destabilize the closed ternary complex so that incorporation of incorrect dNTP would be a rare event.

Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH.

The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2',3'-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 Å resolution) shows two Mn(2+) ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn(2+) complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn(2+) active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3'-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5'-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.

Using a fluorescent cytosine analogue tC(o) to probe the effect of the Y567 to Ala substitution on the preinsertion steps of dNMP incorporation by RB69 DNA polymerase.

Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC(o)), to identify preinsertion step(s) altered by NBP flexibility. When tC(o) is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC(o) is also quenched in the presence of dATP. We determined the crystal structure of the dATP/tC(o)-containing ternary complex of the RB69pol Y567A mutant at 1.9 Å resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC(o). Stabilization of the dATP/tC(o) base pair involved movement of the tC(o) backbone sugar into the DNA minor groove and required tilting of the tC(o) tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity.

Contribution of partial charge interactions and base stacking to the efficiency of primer extension at and beyond abasic sites in DNA.

During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

Probing minor groove hydrogen bonding interactions between RB69 DNA polymerase and DNA.

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 10(2)-10(3)-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 10(2)-10(3)-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.

Structural basis for differential insertion kinetics of dNMPs opposite a difluorotoluene nucleotide residue.

We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP insertion opposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-state kinetic parameters for incorporation of four dNMPs opposite dF by RB69pol and solved the structures of corresponding ternary complexes. We observed that both the F2 and F4 substituent of dF in these structures serve as HB acceptors forming HBs either directly with dTTP and dGTP or indirectly with dATP and dCTP via ordered water molecules. We have defined the shape and chemical features of each dF/dNTP pair in the RB69pol active site without the corresponding phosphodiester-linkage constraints of dF/dNs when they are embedded in isolated DNA duplexes. These features can explain the kinetic preferences exhibited by the templating dF when the nucleotide incorporation is catalyzed by wild type RB69pol or its mutants. We further show that the shapes of the dNTP/dF nascent base pair differ markedly from the corresponding dNTP/dT in the pol active site and that these differences have a profound effect on their incorporation efficiencies.

Bidentate and tridentate metal-ion coordination states within ternary complexes of RB69 DNA polymerase.

Two divalent metal ions are required for primer-extension catalyzed by DNA polymerases. One metal ion brings the 3'-hydroxyl of the primer terminus and the α-phosphorus atom of incoming dNTP together for bond formation so that the catalytically relevant conformation of the triphosphate tail of the dNTP is in an α,ß,γ-tridentate coordination complex with the second metal ion required for proper substrate alignment. A probable base selectivity mechanism derived from structural studies on Dpo4 suggests that the inability of mispaired dNTPs to form a substrate-aligned, tridentate coordination complex could effectively cause the mispaired dNTPs to be rejected before catalysis. Nevertheless, we found that mispaired dNTPs can actually form a properly aligned tridentate coordination complex. However, complementary dNTPs occasionally form misaligned complexes with mutant RB69 DNA polymerases (RB69pols) that are not in a tridentate coordination state. Here, we report finding a ß,γ-bidentate coordination complex that contained the complementary dUpNpp opposite dA in the structure of a ternary complex formed by the wild type RB69pol at 1.88 Å resolution. Our observations suggest that several distinct metal-ion coordination states can exist at the ground state in the polymerase active site and that base selectivity is unlikely to be based on metal-ion coordination alone.

Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase.

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,ß-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.