Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.     

On the metabolic activation of the carcinogen N-hydroxy-N-2-acetylaminofluorene : III. Oxidation with horseradish peroxidase to yield 2-nitrosofluorene and N-acetoxy-N-2-acetylaminofluorene

Previous studies have shown that the carcinogen N-hydroxy-2-acetylaminofluorene is converted by one-electron oxidants to a free nitroxide radical which dismutates to N-acetoxy-2-acetylaminofluorene and 2-nitrosofluorene. The present study shows that the same oxidation can be achieved with horseradish peroxidase and H₂O₂. The free radical intermediate was detected by its ESR signal, and the yields of N-acetoxy-2-acetylaminofluorene and of 2-nitrosofluorene were determined under a number of conditions. Addition of tRNA to the reaction mixture containing N-acetoxy-N-2-acetyl[2′-³H]aminofluorene yielded tRNA-bound radioactivity; addition of guanosine yielded a reaction product which appears to be N-guanosin-8-yl)-2-acetylaminofluorene. The latter compound has previously been identified as a reaction product of N-acetoxy-2-acetylaminofluorene and guanosine. Preliminary attempts to demonstrate the formation of a nitroxide free radical or its dismutation products with rat liver mixed function oxidase systems were not successful.     

Functional Importance of Bacteriophage ?29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

Recent crystallographic resolution of ?29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx₂h motif of proofreading family B DNA polymerases. Single substitution of ?29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx₂hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at ?29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.     

Effect of N-Guanyl Modifications on Early Steps in Catalysis of Polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N²-alkyl guanine (G) adducts with N²-isobutylG showing the largest effect, decreasing ∼ 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N²-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2′-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N²-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N²-alkylG adducts. With the smaller N²-alkylG adducts, the conformational rate constants were not changed dramatically (<  3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (≥  (2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

A new anti conformation for N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) allows Watson-Crick pairing in the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)

Primer extension studies have shown that the Y-family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus P2 can preferentially insert C opposite N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) [F. Boudsocq, S. Iwai, F. Hanaoka and R. Woodgate (2001) Nucleic Acids Res., 29, 4607–4616]. Our goal is to elucidate on a structural level how AAF-dG can be harbored in the Dpo4 active site opposite an incoming dCTP, using molecular modeling and molecular dynamics simulations, since AAF-dG prefers the syn glycosidic torsion. Both anti and syn conformations of the templating AAF-dG in a Dpo4 ternary complex were investigated. All four dNTPs were studied. We found that an anti glycosidic torsion with C1′-exo deoxyribose conformation allows AAF-dG to be Watson–Crick hydrogen-bonded with dCTP with modest polymerase perturbation, but other nucleotides are more distorting. The AAF is situated in the Dpo4 major groove open pocket with fluorenyl rings 3′- and acetyl 5′-directed along the modified strand, irrespective of dNTP. With AAF-dG syn, the fluorenyl rings are in the small minor groove pocket and the active site region is highly distorted. The anti-AAF-dG conformation with C1′-exo sugar pucker can explain the preferential incorporation of dC by Dpo4. Possible relevance of our new major groove structure for AAF-dG to other polymerases, lesion repair and solution conformations are discussed.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

Aromatic N-oxide bridged copper(II) coordination polymers: Synthesis, characterization and magnetic properties

The complexes [Cu₂(o-NO₂-C₆H₄COO)₄(PNO)₂] (1), [Cu₂(C₆H₅COO)₄(2,2′-BPNO)]_n (2), [Cu₂(C₆H₅COO)₄(4,4′-BPNO)]_n (3), [Cu(p-OH-C₆H₄COO)₂(4,4′-BPNO)₂·H₂O]_n (4), (where PNO = pyridine N-oxide, 2,2′-BPNO = 2,2′-bipyridyl-N,N′-dioxide, 4,4′-BPNO = 4,4′-bipyridyl-N,N′-dioxide) are prepared and characterized and their magnetic properties are studied as a function of temperature. Complex 1 is a discrete dinuclear complex while complexes 2-4 are polymeric of which 2 and 3 have paddle wheel repeating units. Magnetic susceptibility measurements from polycrystalline samples of 1-4 revealed strong antiferromagnetic interactions within the {Cu₂}⁴⁺ paddle wheel units and no discernible interactions between the units. The complex 5, [Cu(NicoNO)₂·2H₂O]_n·4nH₂O, in which the bridging ligand to the adjacent copper(II) ions is nicotinate N-oxide (NicoNO) the transmitted interaction is very weakly antiferromagnetic.     

Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases

Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2′,3′-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion.     

Synthesis, structure, cytotoxic activities and DNA-binding properties of tetracopper(II) complexes with dissymmetrical N,N′-bis(substituted)oxamides as ligands

To compare the cytotoxicities and the DNA-binding properties in tetranuclear complexes with different bridging ligands, two tetracopper(II) complexes with formulae of [Cu₄(oxbe)₂Cl₂(bpy)₂]·4H₂O (1) and [Cu₄(oxbm)₂Cl₂(bpy)₂]·2H₂O (2) were synthesized, where H₃oxbe and H₃oxbm stand for N-benzoato-N′-(2-aminoethyl)oxamide and N-benzoato-N′-(1,2-propanediamine)oxamide, respectively, and bpy is 2,2′-bipyridine. Complex 1 was characterized by elemental analyses, IR and electronic spectra and single-crystal X-ray diffraction. The crystal structure reveals the presence of the circular tetranuclear copper(II) cations which are assembled by a pair of cis-oxamido-bridged dinuclear copper(II) units through carboxyl bridges. The crystal structure of complex 2 has been reported in our previous paper. However, the bioactivities were not studied. Cytotoxicities experiments reveal that both the two complexes exhibit cytotoxic effects against human hepatocellular carcinoma cell SMMC-7721 and human lung adenocarcinoma cell A549, and complex 1 has the better activities than those of complex 2. The results of the interactions between the two complexes and herring sperm DNA (HS-DNA) suggest that the two complexes interact with HS-DNA in the mode of intercalation with the intrinsic binding constants of 3.93 × 10⁴ M⁻¹ (1) and 2.48 × 10⁴ M⁻¹ (2). These results indicated that the bridging ligands may play an important role in the cytotoxicities and the DNA-binding properties of tetranuclear complexes.     

Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases

Chronic inflammation involving constant generation of nitric oxide (NO) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2′-deoxyinosine (dI) and 2′-deoxyxanthosine are primarily formed by NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol α was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol η and pol κΔC (a truncated form of pol κ) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol α, pol η, and pol κΔC, promoted preferential incorporation of 2′-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2′-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol α, pol η, and pol κΔC indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A → G transition. This NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Steric and Electrostatic Effects at the C2 Atom Substituent Influence Replication and Miscoding of the DNA Deamination Product Deoxyxanthosine and Analogs by DNA Polymerases

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T_m and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T_m further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.     

Interplay among replicative and specialized DNA polymerases determines failure or success of translesion synthesis pathways

Living cells possess a panel of specialized DNA polymerases that deal with the large diversity of DNA lesions that occur in their genomes. How specialized DNA polymerases gain access to the replication intermediate in the vicinity of the lesion is unknown. Using a model system in which a single replication blocking lesion can be bypassed concurrently by two pathways that leave distinct molecular signatures, we analyzed the complex interplay among replicative and specialized DNA polymerases. The system involves a single N-2-acetylaminofluorene guanine adduct within the NarI frameshift hot spot that can be bypassed concurrently by Pol II or Pol V, yielding a −2 frameshift or an error-free bypass product, respectively. Reconstitution of the two pathways using purified DNA polymerases Pol III, Pol II and Pol V and a set of essential accessory factors was achieved under conditions that recapitulate the known in vivo requirements. With this approach, we have identified the key replication intermediates that are used preferentially by Pol II and Pol V, respectively. Using single-hit conditions, we show that the β-clamp is critical by increasing the processivity of Pol II during elongation of the slipped −2 frameshift intermediate by one nucleotide which, surprisingly, is enough to support subsequent elongation by Pol III rather than degradation. Finally, the proofreading activity of the replicative polymerase prevents the formation of a Pol II-mediated −1 frameshift product. In conclusion, failure or success of TLS pathways appears to be the net result of a complex interplay among DNA polymerases and accessory factors.     

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase η

N⁶-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (k_pol) and increased the dissociation constant (K_d,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication.     

Effect of oxidatively damaged DNA on the active site preorganization during nucleotide incorporation in a high fidelity polymerase from Bacillus stearothermophilus

We study the effect of the oxidative lesion 8-oxoguanine (8oxoG) on the preorganization of the active site for DNA replication in the closed (active) state of the Bacillus fragment (BF), a Klenow analog from Bacillus stearothermophilus. Our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of BF bound to correct dCTP/incorrect dATP opposite guanine (G) and 8oxoG bases in DNA suggest that the lesion introduces structural and energetic changes at the catalytic site to favor dATP insertion. Despite the formation of a stable Watson-Crick pairing in the 8oxoG:dCTP system, the catalytic geometry is severely distorted to possibly slow down catalysis. Indeed, our calculated free energy landscapes associated with active site preorganization suggest additional barriers to assemble an efficient catalytic site, which need to be overcome during dCTP incorporation opposite 8oxoG relative to that opposite undamaged G. In contrast, the catalytic geometry for the Hoogsteen pairing in the 8oxoG:dATP system is highly organized and poised for efficient nucleotide incorporation via the "two-metal-ion" catalyzed phosphoryl transfer mechanism. However, the free energy calculations suggest that the catalytic geometry during dATP incorporation opposite 8oxoG is considerably less plastic than that during dCTP incorporation opposite G despite a very similar, well organized catalytic site for both systems. A correlation analysis of the dynamics trajectories suggests the presence of significant coupling between motions of the polymerase fingers and the primary distance for nucleophilic attack (i.e., between the terminal primer O3' and the dNTP P(alpha.) atoms) during correct dCTP incorporation opposite undamaged G. This coupling is shown to be disrupted during nucleotide incorporation by the polymerase with oxidatively damaged DNA/dNTP substrates. We also suggest that the lesion affects DNA interactions with key polymerase residues, thereby affecting the enzymes ability to discriminate against non-complementary DNA/dNTP substrates. Taken together, our results provide a unified structural, energetic, and dynamic platform to rationalize experimentally observed relative nucleotide incorporation rates for correct dCTP/incorrect dATP insertion opposite an undamaged/oxidatively damaged template G by BF.     

Structural and functional interaction of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone,a photoreactive bupropion derivative,with nicotinic acetylcholine receptors

The pharmacological properties of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13′) and serine (position 6′) rings. However, an additional domain, between the outer (position 20′) and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.     

X-ray and NMR study of the fate of the Co(1,10-phenanthroline-5,6-diketone)3 ion in aqueous solution: supramolecular motifs in the packing of 1,10-phenanthroline-5,6-diketone and 1,10-phenanthroline-5,6-diol complexes

X-ray structures are presented of three new cobalt complexes prepared from Co(III) and N,N^′-1,10-phenanthroline-5,6-dione. The cis-aqua-chloro-bis(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(II) nitrate trihydrate (3) and the cis-aqua-bromo-bis(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(II) trifluoro-methanesulfonate tetrahydrate (4), crystalize in the same space group with a similar arrangement of the complex ions. However, on the molecular scale there are important differences. The cobalt complex in 3 has a typical high-spin geometry whereas in 4 the cobalt complex displays a Jahn-Teller distortion characteristic for low-spin compounds. The third structure is di(N,N^′-1,10-phenanthroline-5,6-diol)(N,N^′-1,10-phenanthroline-5,6-dione)cobalt(III) bromide hexahydrate (5). NMR studies of the hydration of the Co(III)(1,10-phenanthroline-5,6-dione)₃ ³⁺ ion in water and DMSO are also presented. The various possible transformations of the N,N^′-1,10-phenanthroline-5,6-dione ligand are discussed.