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Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg 2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg 2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg 2+ and the primer 3'-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Helicobacter pylori deoxyuridine triphosphate nucleotidohydrolase (HpdUTPase) is a key enzyme in the synthesis of the thymidine nucleotide pathway. It catalyzes the hydrolysis of dUTP to dUMP and releases pyrophosphate. This enzyme has been shown to be essential in several pathogenic organisms. Here, we have determined the crystal structures of HpdUTPase in complex with α, β-imido dUTP (non-hydrolyzable substrate analog) and apo-state at resolution of 2 Å and 2.5 Å respectively. The flexible c terminal end of HpdUTPase which is not observed in apo-state structure and becomes ordered in the complex structure, suggesting its role in forming active site and substrate interaction. The Isothermal titration calorimetry (ITC) experiments reveal that hydrolysis of dUTP is an exothermic reaction with K m  = 35.0 ± 0.19 μM and the k cat  = 1.20 ± 0.19 s -1 . The ITC studies combined with MD simulations for all other nucleotides (dATP.dGTP, dCTP and dTTP) show that the active site of HpdUTPase strangely can also accommodate dATP. The structural comparison with the host (human) dUTPases reveals critical differences in substrate binding affinity of the active site of HpdUTPase. The detailed study suggests that the dATP binds in the active site of HpdUTPase making the number of preferable hydrogen bonds and shows activity with Km of 47 ± 2.4 μM., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Samudrala Gourinath reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are inhibited from performing motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state can pathologically disrupt IHM structure and the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analog called 2'-deoxy-ATP (dATP) is a potent myosin activator that destabilizes the IHM. Here, we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations of the IHM state containing ADP.Pi in both nucleotide binding pockets revealed dynamic motions of the blocked head-free head interface, light chain binding domain, and S2 in this "inactive" state of myosin. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that increased heterogeneity among residue contact pairs at the blocked head-free head interface and a 14% decrease in the interaction energy at the interface. Dynamic changes to this interface were accompanied by dynamics in the light chain binding region. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β 2 and α 2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β 2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β 2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode ( Caenorhabditis elegans ). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo .

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum , which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Martínez-Carranza et al.)

Telomerase is a specialized reverse transcriptase that adds GGTTAG repeats to chromosome ends and is upregulated in most human cancers to enable limitless proliferation. Here, we uncover two distinct mechanisms by which naturally occurring oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation. We conduct a series of direct telomerase extension assays in the presence of modified dNTPs on various telomeric substrates. We provide direct evidence that telomerase can add the nucleotide reverse transcriptase inhibitors ddITP and AZT-TP to the telomeric end, causing chain termination. In contrast, telomerase continues elongation after inserting oxidized 2-OH-dATP or therapeutic 6-thio-dGTP, but insertion disrupts translocation and inhibits further repeat addition. Kinetics reveal that telomerase poorly selects against 6-thio-dGTP, inserting with similar catalytic efficiency as dGTP. Furthermore, telomerase processivity factor POT1-TPP1 fails to restore processive elongation in the presence of inhibitory dNTPs. These findings reveal mechanisms for targeting telomerase with modified dNTPs in cancer therapy.

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90-120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Atmospheric and room-temperature plasma (ARTP) has been successfully developed as a useful mutation tool for mutation breeding of various microbes and plants as well animals by genetic alterations. However, understanding of the molecular mechanisms underlying the biological responses to ARTP irradiation is still limited. Therefore, to gain a molecular understanding of how irradiation with ARTP damages DNA, we irradiated the artificially synthesized mononucleotides of dATP, dTTP, dGTP, and dCTP, and the oligonucleotides of dA 8 , dT 8 , dG 8 , dC 8 , and dA 2 dT 2 dG 2 dC 2 as chemical building blocks of DNA with ARTP for 1-4 min, identified the mononucleotide products using 31 P- and 1 H-nuclear magnetic resonance spectroscopy (NMR), and identified the oligonucleotide products using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) during ARTP treatment. The observed 31 P-and 1 H-NMR spectrum signals for the plasma-treated and untreated mononucleotides indicated that dATP was less stable to plasma irradiation than the other mononucleotides. The oligonucleotides after treatment with ARTP were found to have been broken into small fragments as shown by mass spectrometry, with the cleaved bonds and produced fragments identified according to their expected spectral m/z values or molecular weights derived from their m/z values. The stabilities of the oligonucleotides differed to ARTP irradiation, with dT 8 being the most stable and was more beneficial to stabilizing single-stranded oligonucleotide structures compared to the other base groups (A, G, and C). This was consistent with the average potential energy level obtained by the molecular dynamic simulation of the oligonucleotides, i.e., dT 8  > dC 8  > dA 8  > dG 8  > dA 2 dT 2 dG 2 dC 2 . In summary, we found that ARTP treatment caused various structural changes to the oligonucleotides that may account for the wide and successful applications reported for ARTP-induced mutation breeding of various organisms.

A unique member of the family of cobalamin (Cbl)-dependent radical S -adenosylmethionine (SAM) enzymes, OxsB, catalyzes the ring constriction of deoxyadenosine triphosphate (dATP) to the base oxetane aldehyde phosphate, a crucial precursor for oxetanocin A (OXT-A), which is an antitumor, antiviral, and antibacterial compound. This enzyme reveals a new catalytic function for this big family that is different from the common methylation. On the basis of density functional theory calculations, a mechanism has been proposed to mainly include that the generation of 5'-deoxyadenosine radical, a hydrogen transfer forming 2'-dATP radical, and a Cbl-catalyzed ring contraction of the deoxyribose in 2'-dATP radical. The ring contraction is a concerted rearrangement step accompanied by an electron transfer from the deoxyribose hydroxyl oxygen to Co III without any ring-opening intermediate. Co II Cbl has been ruled out as an active state. Other mechanistic characteristics are also revealed. This unprecedented non-methylation mechanism provides a new catalytic repertoire for the family of radical SAM enzymes, representing a new class of ring-contraction enzymes.

2-Formyl-2'-deoxyadenosine triphosphate (d CHO ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of d CHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The d CHO ATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo , as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway., (© 2020 Scaletti et al.)

Low-energy (3-25 eV) electron interactions with multilayers of 2'-deoxyadenosine 5'-monophosphate (dAMP) were probed using X-ray photoelectron spectroscopy (XPS). Understanding how electrons damage the nucleotide dAMP, which is a building block of DNA, can give insight into how the DNA undergoes radiation damage. Chemical modifications to the constituent units of the nucleotide were revealed in situ through monitoring of the O 1s, C 1s, and N 1s elemental transitions. It is shown that direct electron irradiation causes decomposition of both the base and sugar subunits, as well as cleavage of glycosidic and phosphoester bonds. Incident electrons undergo inelastic energy losses, including creation of core-excited resonances above 3-4 eV. In the condensed phase, these resonances decay via autoionization, producing electronically excited targets and <3 eV electrons. The excited states dissociate and the slow (<3 eV) electrons are captured by neighboring molecules, forming molecular shape resonances that can lead to bond rupture. Since the observed chemical changes were similar at all incident electron energies studied, they can be primarily attributed to formation and decay of transient negative ions. Damage enhancements in the energy ranges typical of all scattering resonances are expected, with the damage probability dominated by the low-energy shape resonances.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 10 6 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

Influenza A viruses leading to infectious respiratory diseases cause seasonal epidemics and sometimes periodic global pandemics. Viral polymerase is an attractive target in inhibiting viral replication, and 4'-ethynyladenosine, which has been reported as a highly potent anti-human immunodeficiency virus (HIV) nucleoside derivative, can work as an anti-influenza agent. Herein, we designed and synthesized a 4'-ethynyl-2'-deoxyadenosine 5'-monophosphate analog called EdAP ( 5 ). EdAP exhibited potent inhibition against influenza virus multiplication in Madin-Darby canine kidney (MDCK) cells transfected with human α2-6-sialyltransferase (SIAT1) cDNA and did not show any toxicity toward the cells. Surprisingly, this DNA-type nucleic acid analog ( 5 ) inhibited the multiplication of influenza A virus, although influenza virus is an RNA virus that does not generate DNA.

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2'-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade -1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A series of 2-alkylamino-2'-deoxyadenosine triphosphates (dATP) was prepared and found to be substrates for the Therminator DNA polymerase, which incorporated only one modified nucleotide into the primer. Using a template encoding for two consecutive adenines, conditions were found for incorporation of either one or two modified nucleotides. In all cases, addition of a mixture of natural dNTPs led to primer extension resulting in site-specific single modification of DNA in the minor groove. The allylamino-substituted DNA was used for the thiol-ene addition, whereas the propargylamino-DNA for the CuAAC click reaction was used to label the DNA with a fluorescent dye in the minor groove. The approach was used to construct FRET probes for detection of oligonucleotides., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme's quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the α subunit of this RNR contains tightly bound deoxyadenosine 5'-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α 2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α 2 dimer is the predominant form of the dAMP-bound α in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α 2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Synthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution. We discuss possible mechanistic implications of this expanded two-metal-ion architecture, including inferences on medium-resolution cryoelectron microscopy structures. Ultimately, our analysis may inspire future experiments and strategies for enzyme engineering or drug design to modulate nucleic acid processing., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

DNA polymerase (pol) processivity, i.e., the bases a polymerase extends before falling off the DNA, and activity are important for copying difficult DNA sequences, including simple repeats. Y-family pols would be appealing for copying difficult DNA and incorporating non-natural dNTPs, due to their low fidelity and loose active site, but are limited by poor processivity and activity. In this study, the binding between Dbh and DNA was investigated to better understand how to rationally design enhanced processivity in a Y-family pol. Guided by structural simulation, a fused pol Sdbh with non-specific dsDNA binding protein Sso7d in the N-terminus was designed. This modification increased in vitro processivity 4-fold as compared to the wild-type Dbh. Additionally, bioinformatics was used to identify amino acid mutations that would increase stabilization of Dbh bound to DNA. The variant SdbhM76I further improved the processivity of Dbh by 10 fold. The variant SdbhKSKIP241-245RVRKS showed higher activity than Dbh on the incorporation of dCTP (correct) and dATP (incorrect) opposite the G (normal) or 8-oxoG(damaged) template base. These results demonstrate the capability to rationally design increases in pol processivity and catalytic efficiency through computational DNA binding predictions and the addition of non-specific DNA binding domains.

The encapsulation mechanism of DNA in sol-gel derived silica has been explored in order to elucidate the effect of DNA conformation on encapsulation and to identify the nature of chemical/physical interaction of DNA with silica during and after sol-gel transition. In this respect, double stranded DNA and dAMP (2'-deoxyadenosine 5'-monophosphate) were encapsulated in silica using an alkoxide-based sol-gel route. Biomolecule-encapsulating gels have been characterized using UV-Vis, 29 Si NMR, FTIR spectroscopy and gas adsorption (BET) to investigate chemical interactions of biomolecules with the porous silica network and to examine the extent of sol-gel reactions upon encapsulation. Ethidium bromide intercalation and leach out tests showed that helix conformation of DNA was preserved after encapsulation. For both biomolecules, high water-to-alkoxide ratio promoted water-producing condensation and prevented alcoholic denaturation. NMR and FTIR analyses confirmed high hydraulic reactivity (water adsorption) for more silanol groups-containing DNA and dAMP encapsulated gels than plain silica gel. No chemical binding/interaction occurred between biomolecules and silica network. DNA and dAMP encapsulated silica gelled faster than plain silica due to basic nature of DNA or dAMP containing buffer solutions. DNA was not released from silica gels to aqueous environment up to 9 days. The chemical association between DNA/dAMP and silica host was through phosphate groups and molecular water attached to silanols, acting as a barrier around biomolecules. The helix morphology was found not to be essential for such interaction. BET analyses showed that interconnected, inkbottle-shaped mesoporous silica network was condensed around DNA and dAMP molecules.

The human sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a retroviral restriction factor in myeloid cells and non-cycling CD4+ T cells, a feature imputed to its phosphohydrolase activity-the enzyme depletes the cellular dNTP levels inhibiting reverse transcription. The functionally active form of SAMHD1 is an allosterically triggered tetramer which utilizes GTP-Mg +2 -dNTP cross bridges to link and stabilize adjacent monomers. However, very little is known about how it assembles into a tetramer and how long the tetramer stays intact. In this computational study, we provide a molecular dynamics based analysis of the structural stability and allosteric site dynamics in SAMHD1. We have investigated the allosteric links which assemble and hold the tetramer together. We have also extended this analysis to a regulatory mutant of SAMHD1. Experimental studies have indicated that phosphorylation of T592 downregulates HIV-1 restriction. A similar result is also achieved by a phosphomimetic mutation T592E. While a mechanistic understanding of the process is still elusive, the loss of structural integrity of the enzyme is conjectured to be the cause of the impaired dNTPase activity of the T592E mutant. MD simulations show that the T592E mutation causes slightly elevated local motions which remain confined to the short helix (residues 591-595), which contains the phosphorylation site and do not cause long-range destabilization of the SAMHD1 tetramer within the timeframe of the simulations. Thus, the regulatory mechanism of SAMHD1 is a more subtle mechanism than has been previously suspected. Proteins 2017; 85:1266-1275. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine-thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation-π, and π-π interactions of the side chains with the dATP and the TTD or thymine-thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment.

DNA can be damaged by many compounds in our environment, and the resulting damaged DNA is commonly replicated by translesion synthesis (TLS) polymerases. Because the mechanism and efficiency of TLS are affected by the type of DNA damage, obtaining information for a variety of DNA adducts is critical. However, there is no structural information for the insertion of a dNTP opposite an O6-dG adduct, which is a particularly harmful class of DNA lesions. We used molecular dynamics (MD) simulations to investigate structural and energetic parameters that dictate preferred dNTP insertion opposite O6-benzyl-guanine (Bz-dG) by DNA polymerase IV, a prototypical TLS polymerase. Specifically, MD simulations were completed on all possible ternary insertion complexes and ternary -1 base deletion complexes with different Bz-dG conformations. Our data suggests that the purines are unlikely to be inserted opposite anti- or syn-Bz-dG, and dTTP is unlikely to be inserted opposite syn-Bz-dG, because of changes in the active site conformation, including critical hydrogen-bonding interactions and/or reaction-ready parameters compared to natural dG replication. In contrast, a preserved active site conformation suggests that dCTP can be inserted opposite either anti- or syn-Bz-dG and dTTP can be inserted opposite anti-Bz-dG. This is the first structural explanation for the experimentally observed preferential insertion of dCTP and misincorporation of dTTP opposite Bz-dG. Furthermore, we provide atomic level insight into why Bz-dG replication does not lead to deletion mutations, which is in contrast with the replication outcomes of other adducts. These findings provide a basis for understanding the replication of related O6-dG adducts.

β-Cyclodextrin (β-CD), which resides in the α-hemolysin (αHL) protein pore, can act as a molecular adapter in single-molecule exonuclease DNA sequencing approaches, where the different nucleotide binding behavior of β-CD is crucial for base discrimination. In the present contribution, the inclusion modes of β-CD towards four 2'-deoxyribonucleoside 5'-monophosphates (dNMPs) were investigated using quantum mechanics (QM) calculations. The calculated binding energy suggests that the binding affinity of dAMP to β-CD are highest among all the dNMPs in solution, in agreement with experimental results. Geometry analysis shows that β-CD in the dAMP complex undergoes a small conformational change, and weak interaction analysis indicates that there are small steric repulsion regions in β-CD. These results suggest that β-CD has lower geometric deformation energy in complexation with dAMP. Furthermore, topological analysis and weak interaction analysis suggest that the number and strength of intermolecular hydrogen bonds and van der Waals interactions are critical to dAMP binding, and they both make favorable contributions to the lower interaction energy. This work reveals the reason why β-CD prefers to bind dAMP rather than other dNMPs, while opening exciting perspectives for the design of novel β-CD-based molecular adapters in the single-molecule exonuclease method of sequencing DNA. Graphical Abstract The binding affinity of β-cyclodextrin towards four 2'-deoxyribonucleoside 5'-monophosphates was investigated using quantum mechanics calculations.

Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2-deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross-bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we found that dATP increased motility. These results highlight alterations to myosin that enhance cross-bridge formation and reveal a potential mechanism that may underlie dATP-induced improvements in cardiac function., (© 2017 The Protein Society.)

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH 2 , CH 3 , vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat /K m values >10,000 M -1  s -1 : Q92EH0&#95;LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1&#95;BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5&#95;CLOP1 and Q0TS82&#95;CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat /K m values exhibited against these canonical substrates are well under 10 5 M -1  s -1 . By contrast, several Nudix enzymes show much larger k cat /K m values (in the range of 10 5 to >10 7 M -1  s -1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Additions of alkyl- or arylthiols to 7-vinyl-7-deaza-2'-deoxyadenosine gave a series of 7-[2-(alkyl- or arylsulfanyl)ethyl]-7-deaza-2'-deoxyadenosines in 45-85% yields. The nucleosides were converted to 5'-O-mono-(dA SR MP) or triphosphates (dA SR TP) by phosphorylation. The modified triphosphates were also prepared by thiol addition to 7-vinyl-7-deaza-dATP. The triphosphates dA SR TP were good substrates for DNA polymerases useful in the enzymatic synthesis of base-modified oligonucleotides (ONs) or DNA containing flexibly linked hydrophobic substituents in the major groove. Primer extension was used for the synthesis of ONs with one or several modifications, PCR was used for the synthesis of heavily modified DNA, whereas terminal deoxynucleotidyl transferase was used for a single-nucleotide labeling of the 3'-end.

Four 6-substituted 4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (dA(BX)TPs) were prepared by glycosylation of 4,6-dichloropyrimidoindole followed by ammonolysis, cross-coupling and triphosphorylation. They were found to be moderate to good substrates for DNA polymerases in primer extension. They also exerted fluorescence with emission maxima 335-378nm. When incorporated to oligonucleotide probes, they did not show significant mismatch discrimination but the 6-benzofuryl 4-amino-pyrimido[4,5-b]indole nucleotide displayed a useful sensitivity to protein binding in experiment with SSB protein., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments combined with theoretical calculations are performed to investigate the stable gas-phase conformations of the protonated adenine mononucleotides, [pdAdo+H](+) and [pAdo+H](+). Conformations that are present in the experiments are elucidated via comparative analyses of the experimental IRMPD spectra and the B3LYP/6-311+G(d,p) IR spectra predicted for the conformers optimized at this level of theory. N3 protonation is preferred as it induces base rotation, which allows a strong hydrogen bond to be formed between the excess proton of adenine and the phosphate moiety. In contrast, both N1 and N7 protonation are predicted to be >35 kJ/mol less favorable than N3 protonation. Only N3 protonated conformers are present in the experiments in measurable abundance. Both the low-energy conformers computed and the experimental IRMPD spectra of [pdAdo+H](+) and [pAdo+H](+) indicate that the 2'-hydroxyl moiety does not significantly impact the structure of the most stable conformer or the IRMPD spectral profile of [pAdo+H](+) vs that of [pdAdo+H](+). However, the 2'-hydroxyl leads to a 3-fold enhancement in the IRMPD yield of [pAdo+H](+) in the fingerprint region. Comparison of present results to those reported in a previous IRMPD study of the analogous protonated adenine nucleosides allows the effects of the phosphate moiety on the gas-phase conformations to be elucidated.

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.

In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

The most common lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the "A rule"). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010 J. Mol. Biol. 404, 34-44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5' to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5' to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a "purine rule." A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5' T in the template. We conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl (FABP) or N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5'-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo(-)) or polymerase β (pol β). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo(-) binds strongly to a double strand/single strand template/primer junction, whereas pol β binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol β, Kf-exo(-) binds tightly to lesion-modified templates; however, both polymerases exhibited minimal nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and (19)F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared to that in CG*A. Although Kf-exo(-) was not sensitive to the presence of a DNA lesion, FAAF-induced conformational heterogeneity perturbed the active site of pol β, weakening the enzyme's ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.

In synthesizing a double-stranded DNA from viral RNA, HIV-1 reverse transcriptase (RT) generates an RNA/DNA intermediate. RT also degrades the RNA strand and synthesizes the second DNA strand. The RNase H active site of RT functions as a nuclease to cleave the RNA strand; however, the structural basis for endonucleolytic cleavage of the RNA strand remains elusive. Here we report crystal structures of RT-RNA/DNA-dATP and RT-RNA/DNA-nevirapine (NVP) ternary complexes at 2.5 and 2.9 Å resolution, respectively. The polymerase region of RT-RNA/DNA-dATP complex resembles DNA/DNA ternary complexes apart from additional interactions of 2'-OH groups of the RNA strand. The conformation and binding of RNA/DNA deviates significantly after the seventh nucleotide versus a DNA/DNA substrate. Binding of NVP slides the RNA/DNA non-uniformly over RT, and the RNA strand moves closer to the RNase H active site. Two additional structures, one containing a gapped RNA and another a bulged RNA, reveal that conformational changes of an RNA/DNA and increased interactions with the RNase H domain, including the interaction of a 2'-OH with N474, help to position the RNA nearer to the active site. The structures and existing biochemical data suggest a nucleic acid conformation-induced mechanism for guiding cleavage of the RNA strand., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3-4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent Km for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower Km constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca(++)-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity (VRTF) at both maximal and submaximal Ca(++) levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca(++)-sensitizing mutation in the Ca(++)-binding subunit of Tn (TnC) increased VRTF at submaximal Ca(++) under acidic conditions but had no effect on VRTF at maximal Ca(++) levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca(++). Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases., (Copyright © 2013 Wiley Periodicals, Inc.)

In eukaryotes, the poly(A) tail added at the 3' end of an mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing or post-translational modifications. The complexity of PAP isoforms suggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs are grouped into three clades termed α, β and γ, which originated from two gene duplication events. To date, all the available PAP structures are from the PAPα clade. Here, we present the crystal structure of the first representative of the PAPγ clade, human PAPγ bound to cordycepin triphosphate (3'dATP) and Ca(2+). The structure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservation reveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are more divergent., (© 2013.)

The modified base 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxoG) is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetic data reveal that DNA polymerase X (pol X) from the African swine fever virus incorporates adenine (dATP) opposite to oxoG with higher efficiency than the non-damaged G:C basepair. To help interpret the kinetic data, we perform molecular dynamics simulations of pol X/DNA complexes, in which the template base opposite to the incoming dNTP (dCTP, dATP, dGTP) is oxoG. Our results suggest that pol X accommodates the oxoGsyn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson-Crick complexes. Moreover, for both the oxoGsyn:A and oxoG:C ternary complexes, conformational sampling of the polymerase follows previously described large subdomain movements, local residue motions, and active site reorganization. Interestingly, the oxoGsyn:A system exhibits superior active site geometry in comparison to the oxoG:C system. Simulations for the other mismatch basepair complexes reveal large protein subdomain movement for all systems, except for oxoG:G, which samples conformations close to the open state. In addition, active site geometry and basepairing of the template base with the incoming nucleotide, reveal distortions and misalignments that range from moderate (i.e., oxoG:Asyn) to extreme (i.e., oxoGanti/syn:G). These results agree with the available kinetic data for pol X and provide structural insights regarding the mechanism by which this polymerase can accommodate incoming nucleotides opposite oxoG. Our simulations also support the notion that α-helix E is involved both in DNA binding and active site stabilization. Our proposed mechanism by which pol X can preferentially accommodate dATP opposite template oxoG further underscores the role that enzyme dynamics and conformational sampling operate in polymerase fidelity and function., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Biopterin (Bip) and its photoproducts 6-formylpterin (Fop) and 6-carboxypterin (Cap) accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder where the protection against UV radiation fails because of the lack of melanin. These compounds absorb in the UV-A inducing a potential photosensitizing action that can cause damage to DNA and other biomolecules. In this work, we have investigated the capability of these pterin derivatives (Pt) to act as photosensitizers under UV-A irradiation for the degradation of 2'-deoxyadenosine 5'-monophosphate (5'-dAMP) in aqueous solutions, as model DNA target. Steady-state and time-resolved experiments were performed and the effect of pH was evaluated. The results showed that photosensitized degradation of 5'-dAMP was only observed under acidic conditions, and a mechanistic analysis revealed the participation of the triplet excited state of the pterin derivatives ((3)Pt*) by electron transfer yielding the corresponding pair of radical ions (Pt(•-) and 5'-dAMP(•+)), with successive photosensitizer recovery by electron transfer from Pt(•-) to O2. Finally, 5'-dAMP(•+) participates in subsequent reactions to yield degradation products., (© 2013 The American Society of Photobiology.)

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Background: Conformational selection plays a key role in the polymerase cycle., Results: Klentaq1 exists in conformational equilibrium between three states (open, closed, and “nucleotide-binding”) whose level of occupancy is determined by the bound substrate., Conclusion: The “nucleotide-binding” state plays a pivotal role in the reaction pathway., Significance: Direct evidence is provided for the role of a conformationally distinct “nucleotide-binding” state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

Protecting group chemistry meets molecular biology: Chemically modified dATP carrying a bulky triethylsilylethynyl group was used in a PCR-based synthesis of a gene internally protected against cleavage by restriction endonucleases. The unmodified flanking regions were cleaved for cloning into a plasmid which was replicated by E. coli, and used for protein production., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Y-family DNA polymerase IV or PolIV (Escherichia coli) is the founding member of the DinB family and is known to play an important role in stress-induced mutagenesis. We have determined four crystal structures of this enzyme in its pre-catalytic state in complex with substrate DNA presenting the four possible template nucleotides that are paired with the corresponding incoming nucleotide triphosphates. In all four structures, the Ser42 residue in the active site forms interactions with the base moieties of the incipient Watson-Crick base pair. This residue is located close to the centre of the nascent base pair towards the minor groove. In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala. In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis. Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations.