DNA Polymerase Active Site Research Articles

Study of Inhibition DNA Polymerase by Phenolic Compounds in Traditional Food Spices on the Polymerase Chain Reaction Process

Polymerase Chain Reaction (PCR) is a repeated cycle process including denaturation, annealing and extension using the DNA polymerase enzyme. This identification method is based on specific DNA which increases the number of DNA strands millions of times. The aim of this study was to identify and classify phenolic group compounds in traditional food spices that have the potential to inhibit DNA polymerase and their mechanism of action in the PCR process. The observation made was the Literature Review study as an analytical test method regarding the study of DNA polymerase inhibition by phenolic group compounds in traditional food spices in the PCR process. The results of this study were analyzed from several appropriate literature studies showing that most of the spices in traditional foods contain secondary metabolites of phenolic group compounds. These compounds have been shown to inhibit DNA polymerase in the PCR process, one of the mechanisms is to bind to the active site of DNA polymerase which causes decreased activity and deactivates DNA polymerase. Moreover, it can degrade and denaturate DNA polymerase. This makes it difficult to identify haram substances in processed food products using PCR.

Incorporation of N7-Platinated Guanines into Thermus Aquaticus (Taq) DNA Polymerase: Atomistic Insights from Molecular Dynamics Simulations.

In this work, we elucidated some key aspects of the mechanism of action of the cisplatin anticancer drug, cis-[Pt(NH3)2Cl2], involving direct interactions with free nucleotides. A comprehensive in silico molecular modeling analysis was conducted to compare the interactions of Thermus aquaticus (Taq) DNA polymerase with three distinct N7-platinated deoxyguanosine triphosphates: [Pt(dien)(N7-dGTP)] (1), cis-[Pt(NH3)2Cl(N7-dGTP)] (2), and cis-[Pt(NH3)2(H2O)(N7-dGTP)] (3) {dien = diethylenetriamine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate}, using canonical dGTP as a reference, in the presence of DNA. The goal was to elucidate the binding site interactions between Taq DNA polymerase and the tested nucleotide derivatives, providing valuable atomistic insights. Unbiased molecular dynamics simulations (200 ns for each complex) with explicit water molecules were performed on the four ternary complexes, yielding significant findings that contribute to a better understanding of experimental results. The molecular modeling highlighted the crucial role of a specific α-helix (O-helix) within the fingers subdomain, which facilitates the proper geometry for functional contacts between the incoming nucleotide and the DNA template needed for incorporation into the polymerase. The analysis revealed that complex 1 exhibits a much lower affinity for Taq DNA polymerase than complexes 2-3. The affinities of cisplatin metabolites 2-3 for Taq DNA polymerase were found to be quite similar to those of natural dGTP, resulting in a lower incorporation rate for complex 1 compared to complexes 2-3. These findings could have significant implications for the cisplatin mechanism of action, as the high intracellular availability of free nucleobases might promote the competitive incorporation of platinated nucleotides over direct cisplatin attachment to DNA. The study's insights into the incorporation of platinated nucleotides into the Taq DNA polymerase active site suggest that the role of platinated nucleotides in the cisplatin mechanism of action may have been previously underestimated.

Development of Provesicular Nanodelivery System of Curcumin as a Safe and Effective Antiviral Agent: Statistical Optimization, In Vitro Characterization, and Antiviral Effectiveness.

Curcumin is a natural compound that has many medical applications. However, its low solubility and poor stability could impede its clinical applications. The present study aimed to formulate dry proniosomes to overcome these pitfalls and improve the therapeutic efficacy of Curcumin. Curcumin-loaded proniosomes were fabricated by the slurry method according to 32 factorial design using Design-Expert software to demonstrate the impact of different independent variables on entrapment efficiency (EE%) and % drug released after 12 h (Q12h). The optimized formula (F5) was selected according to the desirability criteria. F5 exhibited good flowability and appeared, after reconstitution, as spherical nanovesicles with EE% of 89.94 ± 2.31% and Q12h of 70.89 ± 1.62%. F5 demonstrated higher stability and a significant enhancement of Q12h than the corresponding niosomes. The docking study investigated the ability of Curcumin to bind effectively with the active site of DNA polymerase of Herpes simplex virus (HSV). The antiviral activity and the safety of F5 were significantly higher than Curcumin. F5 improved the safety of Acyclovir (ACV) and reduced its effective dose that produced a 100% reduction of viral plaques. Proniosomes could be promising stable carriers of Curcumin to be used as a safe and efficient antiviral agent.

Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, β-C-Fapy•dGTP, with DNA polymerase β. The crystallographic snapshots and kinetic data indicate that binding of β-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions β-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of β-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase β has evolved to hinder Fapy•dGTP insertion.

Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

We examine the DNA polymerase β (pol β) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel β,γ-CXY-dNTP analogues, including individual β,γ-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log kpol versus p Ka4 of the leaving group bisphosphonic acids are used to interrogate the effects of the base identity, the dNTP analogue leaving group basicity, and the precise configuration of the C-X atom in R and S stereoisomers on the rate-determining step ( kpol). The dNTP analogues provide a range of leaving group basicity and steric properties by virtue of monohalogen, dihalogen, or methyl substitution at the carbon atom bridging the β,γ-bisphosphonate that mimics the natural pyrophosphate leaving group in dNTPs. Brønsted plot relationships with negative slopes are revealed by the data, as was found for the dGTP and dTTP analogues, consistent with a bond-breaking component to the TS energy. However, greater multiplicity was shown in the linear free energy relationship, revealing an unexpected dependence on the nucleotide base for both A and C. Strong base-dependent perturbations that modulate TS relative to ground-state energies are likely to arise from electrostatic effects on catalysis in the pol active site. Deviations from a uniform linear Brønsted plot relationship are discussed in terms of insights gained from structural features of the prechemistry DNA polymerase active site.

Multisubunit Multiactive Site DNA Polymerase Complexes with Coordinated Activities

Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. However, whether those interactions are stable or stochastic is currently being tested. These enzymatic abilities include high processivities, fidelities and plasticities to ensure faithful genome maintenance in spite of many obstacles to DNA synthesis. Many multisubunit enzyme subcomplexes have been identified within the replisome and most stabilize enzyme complexes on DNA for continued and processive enzymatic action over long stretches of the genome. One of the most well characterized subcomplexes is the elongation complex minimally consisting of the DNA polymerase holoenzyme which includes accessory proteins known to stabilize the association of the polymerase and direct repeated catalysis.In particular, the stability of the DNA polymerase holoenzyme is sensitive to stable secondary DNA structures, protein/DNA complexes, and various DNA damage. High fidelity (HiFi) DNA polymerases in complex with their accessory proteins are responsible for the bulk of genome synthesis, however, most organisms encode multiple separate specialized DNA polymerases to aid in overcoming genomic obstacles with translesion synthesis (TLS). The presence of anywhere from 2 to 18 separate DNA polymerases (depending on the organism) all with identical specificities for a 3′‐OH primer/template DNA creates a complex multiequilibrium within a cell. Moreover, many of these DNA polymerases have been shown to directly or indirectly interact to enhance catalysis. For example, a HiFi trimeric DNA polymerase complex has increased kinetics and processivity explained through dynamic active site switching and exchange. Other direct DNA polymerase interactions allow for coordinated synthesis across DNA lesions through a concerted switch of active sites. Oligomeric DNA polymerase active site coordination will be shown using a variety of presteady‐state kinetic and thermodynamic assays. These and other DNA polymerase interactions and equilibria properties will be put into context and compared across different domains of life to illustrate DNA synthesis mechanisms that maintain a high degree of fidelity and plasticity in spite of a multitude of genomic obstacles.Support or Funding InformationBaylor UniversityAmerican Cancer Society (RSG‐11‐049‐01‐DMC)National Science Foundation (NSF1613534)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of error-free replication across benzo[a]pyrene stereoisomers by Rev1 DNA polymerase

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N2 amino group of guanine to generate four stereoisomeric BP-N2-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N2-dG adducts, namely the 10S (+)-trans-BP-N2-dG, 10R (+)-cis-BP-N2-dG, and 10S ( − )-cis-BP-N2-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N2-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts.

Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues.

Members of the nucleoside analogue class of cancer therapeutics compete with canonical nucleotides to disrupt numerous cellular processes, including nucleotide homeostasis, DNA and RNA synthesis, and nucleotide metabolism. Nucleoside analogues are triphosphorylated and subsequently inserted into genomic DNA, contributing to the efficacy of therapeutic nucleosides in multiple ways. In some cases, the altered base acts as a mutagen, altering the DNA sequence to promote cellular death; in others, insertion of the altered nucleotide triggers DNA repair pathways, which produce lethal levels of cytotoxic intermediates such as single and double stranded DNA breaks. As a prerequisite to many of these biological outcomes, the modified nucleotide must be accommodated in the DNA polymerase active site during nucleotide insertion. Currently, the molecular contacts that mediate DNA polymerase insertion of modified nucleotides remain unknown for multiple therapeutic compounds, despite decades of clinical use. To determine how modified bases are inserted into duplex DNA, we used mammalian DNA polymerase β (pol β) to visualize the structural conformations of four therapeutically relevant modified nucleotides, 6-thio-2'-deoxyguanosine-5'-triphosphate (6-TdGTP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (5-FdUTP), 5-formyl-deoxycytosine-5'-triphosphate (5-FodCTP), and 5-formyl-deoxyuridine-5'-triphosphate (5-FodUTP). Together, the structures reveal a pattern in which the modified nucleotides utilize Watson-Crick base pairing interactions similar to that of unmodified nucleotides. The nucleotide modifications were consistently positioned in the major groove of duplex DNA, accommodated by an open cavity in pol β. These results provide novel information for the rational design of new therapeutic nucleoside analogues and a greater understanding of how modified nucleotides are tolerated by polymerases.

Elaborated Action of the Human Primosome.

The human primosome is a 340-kilodalton complex of primase (DNA-dependent RNA polymerase) and DNA polymerase α, which initiates genome replication by synthesizing chimeric RNA-DNA primers for DNA polymerases δ and ε. Accumulated biochemical and structural data reveal the complex mechanism of concerted primer synthesis by two catalytic centers. First, primase generates an RNA primer through three steps: initiation, consisting of dinucleotide synthesis from two nucleotide triphosphates; elongation, resulting in dinucleotide extension; and termination, owing to primase inhibition by a mature 9-mer primer. Then Polα, which works equally well on DNA:RNA and DNA:DNA double helices, intramolecularly catches the template primed by a 9-mer RNA and extends the primer with dNTPs. All primosome transactions are highly coordinated by autoregulation through the alternating activation/inhibition of the catalytic centers. This coordination is mediated by the small C-terminal domain of the primase accessory subunit, which forms a tight complex with the template:primer, shuttles between the primase and DNA polymerase active sites, and determines their access to the substrate.

The 8-oxo-dGTP interaction with human DNA polymerase β: two patterns of ligand behavior

The behavior of 8-oxo-dGTP molecule in the area of the active site of human DNA polymerase β was investigated using molecular dynamics (MD) calculation. The principle phenomenon revealed as investigation results is existence of two cardinally different models of behavior inherent in 8-oxo-dGTP molecule. In the several cases, 8-oxo-dGTP stably stays in DNA polymerase active site, “keeps in touch” with template nucleotide and maintains the hydrogen bonds with it (stable behavior). In other cases, the ligand molecules lose the connections with template dA and start to migrate inside of enzyme space (migrate behavior). The 8-oxo-dGTP in cases of migrate behavior is still in DNA polymerase space at least over 100 ns and prevents transit of DNA polymerase from closed to open conformation as well as the further binding of incoming dNTP. This observation lets a possibility to consider it as natural inhibitor/regulator of DNA pol β activity.

The Closing Mechanism of DNA Polymerase I at Atomic Resolution

DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl.

Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase

Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged α-helix called the C helix (H594GEIYRRR601), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coli, the wild-type domain (WT) (residues Ile498–Leu671) and two variants that lack the regions containing the C helix (Ile593–Leu603 and Gly595–Thr605, which we called ΔC1 and ΔC2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni2+ affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA–DNA heteroduplex, WT, ΔC1, and ΔC2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24–Leu671) produced 14–20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA–DNA heteroduplex. The activity levels of WT, ΔC1, and ΔC2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain.

Features of the 8-oxo-7,8-dihydro-2′-dGTP behavior in active site of human DNA polymerase β: structural investigation in silico

The oxidized bases in the composition of DNA as well as DNA precursors (desoxynucleotide triphosphates, dNTPs) appearing in living cell as a result of oxidative stress are the one of major sources of genomic instability. Among oxidized forms of nitrogenous bases, the 8-oxo-7,8-dihydro-2-deoxyguanine (8odG, 8-oxo-dG) is the most ubiquitous. This compound has a high mutagenic potential due to its ability to preferably interact with adenine instead of cytosine. In particular, the 8odG in the composition of the incoming nucleotide triphosphate (8-oxo-GTP) is able to immediately incorporate into the growing DNA chain and, thus, to cause the invert replacement dA → dC because it is possible to pair with the incoming dCTP as well as dATP in the next round of DNA replication. The efficiency of 8oxo-dG incorporation in growing DNA clearly depends on the nature of appropriate DNA polymerases. One of the most sensitive to 8-oxo-dGTP is the eukaryotic DNA polymerase β (pol β). The binding of 8-oxo-dGTP in the active center of pol β can result in two different molecular events. First of them is the incorporation of 8oxoguanine into a growing DNA chain, the other is a discrimination of 8-oxo-dGTP from the active center. While effects of incorporation of this modified guanine in DNA are well studied, the immediate consequences of 8-oxo-dGTP discrimination are still unclear. The behavior of 8-oxo-dGTP molecule in the area of the active site of human DNA polymerase β was investigated using molecular dynamics (MD) calculation. The principle phenomenon revealed as investigation results is existence of two cardinally different models of behavior inherent to 8-oxo-dGTP molecule. In two cases the ligand molecules loses the connections with template dA and starts to migrate inside of enzyme space (migrate trajectories). In the other two cases 8-oxo-dGTP stably stays in DNA polymerase active site, “keeps in touch” with template nucleotide and maintains the hydrogen bonds with it (stable trajectories). The spatial structure of 8-oxo-dGTP in stable trajectories appears to be sufficiently rigid despite the presence of number of bonds around which the free rotation is possible, and its conformational energy is characterized by high stability over the time of studied MD. Average values of energy (-10229.7 and -10227.1 kJ/mol) are practically the same for both cases. Amino acid microenvironment of 8-oxo-dGTP also practically doesn’t change over the studied MD interval. Thus, stable variants of 8-oxo-dGTP behavior evidently correspond to case of the further incorporation modified 8-oxo-dG into growing DNA strand. The behavior of 8-oxo-dGTP molecule in migrate trajectories is significantly more complicated. The 8-oxo-dGTP loses the Н-bonds with template dA6 (at 11 and 6.5 ns of MD in first and second case respectively) and starts to migrate in DNA polymerase space. The 8-oxo-dGTP spatial structure regularly exhibits much more flexibility in comparison to itself behavior in stable trajectories that reflects in corresponded values of individual atomic fluctuations. However, contrary to the expectations the general levels of conformational energy of 8-oxo-dGTP as well as energy fluctuation patterns in both migratory trajectories are completely time stable. The average values of conformational energy are -9938.6 and -10018.6 kJ/mol for trajectories 1 and 2 respectively that is slightly more than corresponded values for stable trajectories. The 8-oxo-dGTP movement pathways of don’t coincide each other that is confirmed by differences of their conformational spaces and amino acid microenvironment. It seems to be the most important that 8-oxo-dGTP not only doesn’t leave the enzyme space but directly prevent transition of DNA polymerase from closed to open conformation as well as the further binding of incoming dNTP. This observation lets a possibility to consider it as natural inhibitor of DNA pol β activity and possible intracellular regulator which mediates the direct transition of the cell from normal state to programmed cell death omitting the malignancy stage. Keywords : 8-oxo-7,8-dihydro-2′-dGTP, 8-oxo-dGTP, DNA polymerase β, mole­cular dynamics, structural analysis, delayed dissociation.

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.

Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue

EFV (efavirenz) and β-thujaplicinol [2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one] have contrasting effects on the RNase H activity of HIV-1 RT (reverse transcriptase). EFV binds in the non-nucleoside inhibitor-binding pocket and accelerates this activity, whereas β-thujaplicinol binds in the RNase H active site and inhibits it. We have used pre-steady-state kinetic analyses to gain an insight into the mechanism by which EFV and a β-thujaplicinol analogue [19616 (2,7-dihydroxy-2,4,6-cyclo-heptatrien-1-one)] modulate RT RNase H activity. Our data show that EFV and 19616 have no effect on polymerase-dependent RNase H cleavages. However, both compounds significantly affected the rates of polymerase-independent RNase H cleavages. In regard to the latter, we found no evidence that the bound RNA/DNA template/primer substrate restricted 19616 from interacting with RT. In light of these data, we propose a model in which 19616 binds to the RNase H active site of RT after the primary polymerase-dependent RNase H cleavage has occurred and stabilizes the 3'-end of the DNA primer in the polymerase active site thus blocking the enzyme's ability to carry out the polymerase-independent cleavages. By contrast, EFV destabilizes the 3'-end of the DNA primer in the DNA polymerase active site and promotes RT-mediated polymerase-independent cleavages. Consistent with this model, we show antagonism between EFV and 19616.

Novel insights from structural analysis of lentiviral and gammaretroviral reverse transcriptases in complex with RNA/DNA hybrids

Structures of HIV-1 reverse transcriptase (RT) have been reported in several forms, but only one contains an RNA/DNA hybrid, the conformation of which has been controversial. We have been successful in obtaining three structures of HIV-1 RT complexed with a non-nucleoside RT inhibitor (NNRTI) and an RNA/DNA hybrid [1]. In the presence of an NNRTI, our RNA/DNA structure differs from all prior nucleic acid bound to RT including the previously-reported RNA/DNA hybrid derived froom the polypurine tract. The enzyme structure observed in our cocrystals also differs from all previous RT-DNA complexes. As a result, the hybrid has ready access to the ribonuclease H (RNase H) active site. These observations collectively reinforce previous proposals that an RT-nucleic acid complex may be required to adopt independent structural states competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Structural features of the nucleoprotein complex, including drug resistance mutations, have been verified by site-directed mutagenesis, and will be presented. Although the single-subunit RT of Moloney murine leukemia virus (Mo-MLV) has been extensively characterized biochemically, structural information is lacking that describes the substrate binding mechanism for this RT species. We also present data on the first crystal structure of a complex between an RNA/DNA hybrid and the 72 kDa single-subunit RT from the related xenotropic murine leukemia virus-related virus (XMRV) [2]. A comparison of this structure with its HIV-1 counterpart shows that substrate binding around the DNA polymerase active site is conserved but differs between the two enzymes in their thumb and connection subdomains. Small-angle X-ray scattering (SAXS) was used to model full-length XMRV RT, demonstrating its flexible RNase H domain becomes ordered in the presence of substrate, a key difference between monomeric and dimeric RTs.

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase β (pol β) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

Amino Acid Substitution in the Active Site of DNA Polymerase β Explains the Energy Barrier of the Nucleotidyl Transfer Reaction

DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair, where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3'group, thus activating O3'as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3'proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3' proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3'. The theoretical results with the wild-type enzyme indicate an early charge reorganization associated with the O3' proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.

Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416-Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M', Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on α-M', (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416-Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.