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Insights into base selectivity from the 1.8 A resolution structure of an RB69 DNA polymerase ternary complex.

ABSTRACT: Bacteriophage RB69 DNA polymerase (RB69 pol) has served as a model for investigating how B family polymerases achieve a high level of fidelity during DNA replication. We report here the structure of an RB69 pol ternary complex at 1.8 Å resolution, extending the resolution from our previously reported structure at 2.6 Å [Franklin, M. C., et al. (2001) Cell 105, 657-667]. In the structure presented here, a network of five highly ordered, buried water molecules can be seen to interact with the N3 and O2 atoms in the minor groove of the DNA duplex. This structure reveals how the formation of the closed ternary complex eliminates two ordered water molecules, which are responsible for a kink in helix P in the apo structure. In addition, three pairs of polar-nonpolar interactions have been observed between (i) the Cα hydrogen of G568 and the N3 atom of the dG templating base, (ii) the O5' and C5 atoms of the incoming dCTP, and (iii) the OH group of S565 and the aromatic face of the dG templating base. These interactions are optimized in the dehydrated environment that envelops Watson-Crick nascent base pairs and serve to enhance base selectivity in wild-type RB69 pol.

SUBMITTER: Wang M

PROVIDER: S-EPMC3036992 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structure of New Binary and Ternary DNA Polymerase Complexes From Bacteriophage RB69.

Project description:DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.

| S-EPMC8639217 | biostudies-literature

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

Project description: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.

| S-EPMC3970900 | biostudies-literature

RB69 DNA polymerase mutants with expanded nascent base-pair-binding pockets are highly efficient but have reduced base selectivity.

Project description:We have investigated the effect of systematically enlarging the nascent base-pair-binding pocket (NBP) of a replicative DNA polymerase from bacteriophage RB69 (RB69 pol) on the incorporation efficiency (k(pol)/K(d,app)) for both correct and incorrect dNMPs. Accordingly, we replaced residues L561, Y567, and S565 in the NBP with Ala, Ala, and Gly, respectively. We combined L561A and Y567A to give a double mutant and then introduced the S565G mutation to give a triple mutant. The efficiency of incorrect dNMP insertion increased markedly relative to the wild type with the single mutants and increased further as the number of substitutions in the NBP increased. The difference in incorporation efficiency for mispairs between the mutants and the wild-type RB69 pol was due mainly to k(pol). Unexpectedly, enlarging the NBP had a minimal effect on the incorporation efficiency of correct dNMPs. Our kinetic data suggest that replicative DNA pols exert base discrimination via "negative selection" against mispairs by using residues in the NBP, particularly the three residues analyzed in this study, to allow rapid incorporation of only correct base pairs. This proposal differs from how geometry and "tightness of fit" of the NBP is often invoked to account for rapid incorporation of correct base pairs, namely, that a tighter fit within the NBP leads to an increase in insertion rates [Kool, E. T. (2002) Annu. Rev. Biochem. 71, 191-219]. We related our findings to that of a model translesion DNA pol, Sulfolobus solfataricus Dpo4. We concur with the main conclusion of a previous study [Mizukami, S., et al. (2006) Biochemistry 45, 2772-2778], namely, that lesion bypass pols exhibit low incorporation efficiencies for correct dNMPs (leading to relative promiscuity) not because of a more open NBP but because of a loose fit of substrates bound in the catalytic centers. This is a property not shared by RB69 pol and its mutants.

| S-EPMC2847438 | biostudies-literature

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

Project description: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,β-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.

| S-EPMC3760225 | biostudies-literature

Hydrogen-bonding capability of a templating difluorotoluene nucleotide residue in an RB69 DNA polymerase ternary complex.

Project description:Results obtained using 2,4-difluorotoluene nucleobase (dF) as a nonpolar thymine isostere by Kool and colleagues challenged the Watson-Crick dogma that hydrogen bonds between complementary bases are an absolute requirement for accurate DNA replication. Here, we report crystal structure of an RB69 DNA polymerase L561A/S565G/Y567A triple mutant ternary complex with a templating dF opposite dTTP at 1.8 Å-resolution. In this structure, direct hydrogen bonds were observed between: (i) dF and the incoming dTTP, (ii) dF and residue G568 of the polymerase, and (iii) dF and ordered water molecules surrounding the nascent base pair. Therefore, this structure provides evidence that a templating dF can form novel hydrogen bonds with the incoming dTTP and with the enzyme that differ from those formed with a templating dT.

| S-EPMC3139434 | biostudies-literature

The reopening rate of the fingers domain is a determinant of base selectivity for RB69 DNA polymerase.

Project description:Two divalent metal ions are required for nucleotide incorporation by DNA polymerases. Here we use the bacteriophage RB69 DNA polymerase (RB69 pol) and the metal ion exchange-inert nucleotide analogue rhodium(III) deoxythymidine triphosphate (Rh.dTTP) to investigate the requirements of metal binding to the "A" site and to the "B" site, independently. We show that while binding of a metal ion to the A site is required for the nucleotidyl transfer reaction to occur, this metal binding is insufficient to initiate the prechemistry enzyme isomerization that has been observed with this polymerase. Moreover, we show that binding of a deoxynucleoside triphosphate (dNTP), in the absence of a catalytic metal ion, is sufficient to induce this conformational change. In this report, we also present several lines of evidence (from pulse-chase, rapid chemical quench-flow, and stopped-flow fluorescence experiments) for the reverse rate of the enzyme isomerization, closed to open, of a DNA polymerase complex. The implications of these data for the fidelity of DNA polymerization by RB69 pol are discussed.

| S-EPMC2845987 | biostudies-literature

RB69 DNA polymerase structure, kinetics, and fidelity.

Project description: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.

| S-EPMC4018061 | biostudies-literature

Effect of A and B metal ion site occupancy on conformational changes in an RB69 DNA polymerase ternary complex.

Project description:Rapid chemical quench assays, as well as equilibrium and stopped-flow fluorescence experiments, were performed with an RB69 DNA polymerase (RB69 pol)-primer-template (P/T) complex containing 2-aminopurine (dAP) and a metal exchange-inert Rh(III) derivative of a deoxynucleoside triphosphate (Rh.dTTP). The objective was to determine the effect of catalytic metal ion (A site) occupancy on the affinity of an incoming Rh.dTTP for the RB69 pol-P/T binary complex and on the rate of the conformational change induced by Rh.dTTP binding. With Ca(2+) in the A site, the affinity of the incoming Rh.dTTP for the RB69 pol-P/T binary complex and the conformational change rate can be determined in the absence of chemistry. When Mg(2+) was added to a ternary complex containing Rh.dTTP opposite dAP, the templating base, nucleotidyl transfer occurred, but the rate of product formation was only one-tenth of that found with Mg.dTTP, as determined by rapid chemical quench assays. Rates of conformational change subsequent to formation of a ternary complex, in the absence of chemistry, were estimated from the rate of change in dAP fluorescence with an increase in the Rh.dTTP concentration. We have shown that there is an initial rapid quenching of dAP fluorescence followed by a second phase of dAP quenching, which has nearly the same rate as that of dTMP incorporation, as estimated from rapid chemical quench experiments. We have also demonstrated that the affinity of Rh.dTTP for occupancy of the B metal ion site is dependent on the presence of Ca(2+). However, a saturating Rh.dTTP concentration in the absence of Ca(2+) results in full quenching of dAP fluorescence, whereas a saturating Ca(2+) concentration in the absence of Rh.dTTP gives only partial quenching of dAP fluorescence. The implications of these results for the mechanism of Fingers closing, metal ion binding, and base selectivity are discussed.

| S-EPMC2846627 | biostudies-literature

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase.

Project description:The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

| S-EPMC3228362 | biostudies-literature

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

Project description: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,β,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 β,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.

| S-EPMC3375444 | biostudies-literature

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