Phosphodiester Bond Formation Research Articles

Rapid Time Scale Analysis of T4 DNA Ligase-DNA Binding.

DNA ligases, essential to both in vivo genome integrity and in vitro molecular biology, catalyze phosphodiester bond formation between adjacent 3'-OH and 5'-phosphorylated termini in dsDNA. This reaction requires enzyme self-adenylylation, using ATP or NAD+ as a cofactor, and AMP release concomitant with phosphodiester bond formation. In this study, we present the first fast time scale binding kinetics of T4 DNA ligase to both nicked substrate DNA (nDNA) and product-equivalent non-nicked dsDNA, as well as binding and release kinetics of AMP. The described assays utilized a fluorescein-dT labeled DNA substrate as a reporter for ligase·DNA interactions via stopped-flow fluorescence spectroscopy. The analysis revealed that binding to nDNA by the active adenylylated ligase occurs in two steps, an initial rapid association equilibrium followed by a transition to a second bound state prior to catalysis. Furthermore, the ligase binds and dissociates from nicked and nonsubstrate dsDNA rapidly with initial association affinities on the order of 100 nM regardless of enzyme adenylylation state. DNA binding occurs through a two-step mechanism in all cases, confirming prior proposals of transient binding followed by a transition to a productive ligase·nDNA (Lig·nDNA) conformation but suggesting that weaker nonproductive "closed" complexes are formed as well. These observations demonstrate the mechanistic underpinnings of competitive inhibition by rapid binding of nonsubstrate DNA, and of substrate inhibition by blocking of the self-adenylylation reaction through nick binding by deadenylylated ligase. Our analysis further reveals that product release is not the rate-determining step in turnover.

Structure-activity relationships of the ATP cofactor in ligase-catalysed oligonucleotide polymerisations.

A T4 DNA ligase-catalysed oligonucleotide polymerisation process has been recently developed to enable the incorporation of multiple functional groups throughout a nucleic acid polymer. T4 DNA ligase requires ATP as a cofactor to catalyse phosphodiester bond formation during the polymerisation process. Herein, we describe the structure-activity relationship of ATP within the context of T4 DNA ligase-catalyzed oligonucleotide polymerisation. Using high-throughput sequencing, we study not only the influence of ATP modification on polymerisation efficiency, but also on the fidelity and sequence bias of the polymerisation process.

Structural Insights into the Post-Chemistry Steps of Nucleotide Incorporation Catalyzed by a DNA Polymerase.

DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.1-3 The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PPi) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events. Here, by using the triphosphates of chain-terminating antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP), we structurally reveal the correct sequence of post-chemistry steps during nucleotide incorporation by human DNA polymerase β (hPolβ) and provide a structural basis for PPi release. These post-catalytic structures reveal hPolβ in an open conformation with PPi bound in the active site, thereby strongly suggesting that the reverse conformational changes occur prior to PPi release. The results also help to refine the role of the newly discovered third divalent metal ion for DNA polymerase-catalyzed nucleotide incorporation. Furthermore, a post-chemistry structure of hPolβ in the open conformation, following incorporation of (-)3TC-MP, with a second (-)3TC-TP molecule bound to the active site in the absence of PPi, suggests that nucleotide binding stimulates PPi dissociation and occurs before polymerase translocation. Our structural characterization defines the order of the elusive post-chemistry steps in the canonical mechanism of a DNA polymerase.

Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore.

The latch region of the wild-type α-hemolysin (α-HL) protein channel can be used to distinguish single base modifications in double-stranded DNA (dsDNA) via ion channel measurements upon electrophoretic capture of dsDNA in the vestibule of α-HL. Herein, we investigated the use of the latch region to detect a nick in the phosphodiester DNA backbone. The presence of a nick in the phosphodiester backbone of one strand of the duplex results in a significant increase in both the blockade current and noise level relative to the intact duplex. Differentiation between the nicked and intact duplexes based on blockade current or noise, with near baseline resolution, allows real-time monitoring of the rate of T3-DNA ligase-catalyzed phosphodiester bond formation. Under low ionic strength conditions containing divalent cations and a molecular crowding agent (75 mg mL-1 PEG), the rate of enzyme-catalyzed reaction in the bulk solution was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the α-HL protein channel vestibule. Enzyme kinetic results obtained from the nanopore experiments match those from gel electrophoresis under the same reaction conditions, indicating the α-HL nanopore measurement provides a viable approach for monitoring enzymatic DNA repair activity.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro.

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.

Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction.

DNA polymerases (pols) are sophisticated protein machines operating in the replication, repair and recombination of genetic material in the complex environment of the cell. DNA pol reactions require at least two divalent metal ions for the phosphodiester bond formation. We explore two understudied roles of metals in pol transactions with emphasis on polα, a crucial enzyme in the initiation of DNA synthesis. We present evidence that the combination of many factors, including the structure of the template/primer, the identity of the metal, the metal turnover in the pol active site, and the influence of the concentration of nucleoside triphosphates, affect DNA pol synthesis. On the poly-dT70 template, the increase of Mg(2+) concentration within the range typically used for pol reactions led to the severe loss of the ability of pol to extend DNA primers and led to a decline in DNA product sizes when extending RNA primers, simulating the effect of "counting" of the number of nucleotides in nascent primers by polα. We suggest that a high Mg(2+) concentration promotes the dynamic formation of unconventional DNA structure(s), thus limiting the apparent processivity of the enzyme. Next, we found that Zn(2+) supported robust polα reactions when the concentration of nucleotides was above the concentration of ions; however, there was only one nucleotide incorporation by the Klenow fragment of DNA pol I. Zn(2+) drastically inhibited polα, but had no effect on Klenow, when Mg(2+) was also present. It is possible that Zn(2+) perturbs metal-mediated transactions in pol active site, for example affecting the step of pyrophosphate removal at the end of each pol cycle necessary for continuation of polymerization.

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me(2+) ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. Therefore Me(2+) ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual Φ29 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg(2+) and Mn(2+), which support catalysis, and Ca(2+), which supports deoxynucleoside triphosphate (dNTP) binding but not catalysis. We examined their effects on translocation, dNTP binding, and primer strand transfer between the polymerase and exonuclease sites. All three metals cause a concentration-dependent shift in the translocation equilibrium, predominantly by decreasing the forward translocation rate. Me(2+) also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3'-OH group. Me(2+) modulates the translocation rates but not their response to force, suggesting that Me(2+) does not affect the distance to the transition state of translocation. Absent Me(2+), the primer strand transfer pathway between the polymerase and exonuclease sites displays additional kinetic states not observed at >1 mm Me(2+). Complementary dNTP binding is affected by Me(2+) identity, with Ca(2+) affording the highest affinity, followed by Mn(2+), and then Mg(2+). Both Ca(2+) and Mn(2+) substantially decrease the dNTP dissociation rate relative to Mg(2+), while Ca(2+) also increases the dNTP association rate.

Real-Time Observation of Backtracking by Bacterial RNA Polymerase.

RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simple correlation with the instability of the DNA-RNA hybrid, indicating a more complex dependence of backtracking on DNA template sequence. Experiments with transcription through an abasic site in DNA template or neutravidin bound to biotinylated template strand base illustrated an important role of backtracking in defining how RNAP reacts to such obstacles in the DNA template. The described approach will be a useful tool in deciphering the mechanism of backtracking and in studying factors that affect the backtracking.

Large α‐helical movements observed in ternary crystals of RB69 DNA polymerase following nucleotide incorporation

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.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a Kd for DNA of 9.2 nm, with an off-rate constant of 0.013 s(-1)and an on-rate constant of 14 μm(-1) s(-1). dNTP binding is rapid with Kd values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a Kd value for PPi of 3.7 mm and a maximal rate constant of 11 s(-1). Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s(-1); rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s(-1), for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM.

Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and self-concatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions.

Viewing Human DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography.

One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson-Crick base pair with correct deoxycytidine 5'-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5'-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson-Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg(2+) appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3'-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.

Mechanistic insights into filamentous phage integration in Vibrio cholerae.

Vibrio cholerae, the etiological agent of acute diarrhoeal disease cholera, harbors large numbers of lysogenic filamentous phages, contribute significantly to the host pathogenesis and provide fitness factors to the pathogen that help the bacterium to survive in natural environment. Most of the vibriophage genomes are not equipped with integrase and thus exploit two host-encoded tyrosine recombinases, XerC and XerD, for lysogenic conversion. Integration is site-specific and it occurs at dimer resolution site (dif) of either one or both chromosomes of V. cholerae. Each dif sequence contains two recombinase-binding sequences flanking a central region. The integration follows a sequential strand exchanges between dif and attP sites within a DNA-protein complex consisting of one pair of each recombinase and two DNA fragments. During entire process of recombination, both the DNA components and recombinases of the synaptic complex keep transiently interconnected. Within the context of synaptic complex, both of the actuated enzymes mediate cleavage of phosphodiester bonds. First cleavage generates a phosphotyrosyl-linked recombinase-DNA complex at the recombinase binding sequence and free 5′-hydroxyl end at the first base of the central region. Following the cleavage, the exposed bases with 5′-hydroxyl ends of the central region of dif and attP sites melt from their complementary strands and react with the recombinase-DNA phosphotyrosyl linkage of their recombining partner. Subsequent ligation between dif and attP strands requires complementary base pair interactions at the site of phosphodiester bond formation. Integration mechanism is mostly influenced by the compatibility of dif and attP sequences. dif sites are highly conserved across bacterial phyla. Different phage genomes have different attP sequences; therefore they rely on different mechanisms for integration. Here, I review our current understanding of integration mechanisms used by the vibriophages.

Elucidation of kinetic mechanisms of human translesion DNA polymerase κ using tryptophan mutants.

To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3'-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat , suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol ) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair.

Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme

The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.

Site-specific labeling of RNA at internal ribose hydroxyl groups: terbium-assisted deoxyribozymes at work.

A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb(3+) as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2'-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2',5'-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ~160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA.

Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis

In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn2+ stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF.

The Ins and Outs of Viral RNA Polymerase Translocation

Central to the replication of RNA viruses is the RNA-dependent RNA polymerase (RdRp) [1], an important antiviral drug target [2–4]. RdRps belong to a large superfamily of template-directed nucleic acid polymerases, including DNA polymerases and reverse transcriptases [1]. These polymerases have a similar structural fold, which has been described as a “cupped right hand” with fingers, thumb and palm subdomains [5] (Fig. 1), and a similar chemical mechanism, involving at least two metal ions [6]. Polymerase function can be divided into three phases, including nucleotide selection, phosphodiester bond formation and translocation to the next templating nucleobase to prepare for the next round of nucleotide addition. Structural biology has provided tremendous insight into the structural changes that accompany each stage of catalysis for multiple classes of polymerases [7–10]. Least understood is how conformational changes in the polymerases may couple to translocation. In A-family DNA polymerases, a partial opening of the active site involving the temporary displacement of the “O-helix” and other regions in the fingers subdomain is likely important for movement along the DNA [11– 15]. Such a mechanism is unlikely to aid translocation in RdRps; these polymerases contain an extension of the fingers through the “fingertips” that interacts with the thumb domain, likely precluding a similar “opening” of the active site [16] (Fig. 1). In this issue, Sholders and Peersen have provided tantalizing insight into an alternative mechanism for translocation in RdRps, involving a palm structural motif known as “motif-B” [17]. Steric clashes between the motif-B loop and the template RNA may promote movement along the RNA to the next catalytic register. These findings are especially intriguing considering that the motif-B loop is involved in multiple stages of RdRp catalysis and

Watching the RNA Polymerase Transcription by Time-Dependent Soak-Trigger-Freeze X-Ray Crystallography

The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction. X-ray crystallography can reveal high resolution structures; however, one perceived limitation is that it reveals only static views. Here we use time-dependent soak-trigger-freeze X-ray crystallography, namely, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction and freezing crystals at different time points, to capture real-time intermediates in the pathway of transcription. In each crystal structure, different intensities and shapes of electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site which allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides the temporal order of substrate assembly and metal co-factor binding at the active site of enzyme which completes our understanding of the two-metal-ion mechanism and fidelity mechanism in single-subunit RNAPs. The nucleotide-binding metal (MeB) is coordinated at the active site prior to the catalytic metal (MeA). MeA coordination is only temporal, established just before and dissociated immediately after phosphodiester bond formation. We captured these elusive intermediates exploiting the slow enzymatic reaction in crystallo. These results demonstrate that the simple time-dependent soak-trigger-freeze X-ray crystallography offers a direct means for monitoring enzymatic reactions. [Ref: Basu, R.S., and K.S. Murakami (2013). J Biol Chem., 288, 3305-3311]

Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide

The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding ϕ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg(2+) ions, a reaction highly improved when Mn(2+) was used as metal activator. These results, together with previous crystallographic resolution of ϕ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg(2+). Recovery of both reactions with Mn(2+) supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP.