Distant Neighbor Base Sequence Context Effects in Human Nucleotide Excision Repair of a Benzo[a]pyrene-derived DNA Lesion

The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N²-dG (G?) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G?-C-T-A-C-C-3′ (CG?C-I), and 5′-C-A-C3-A4-C5-G?-C-A-C-A-C-3′ (CG?C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(± 0.2)-fold greater in the case of the CG?C-II than the CG?C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG?C-II duplex is more bent than the CG?C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG?C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG?C-II than in CG?C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG?C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N²-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on ³²P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)–electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [¹³C₁₀]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2′-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8 ± 3.7 PhIP-C8-dG adducts per 10⁶ 2′-deoxynucleosides. The method required 50 μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10⁸ 2′-deoxynucleosides). In summary, the LC–ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.     

3-Arylisoquinolines as novel topoisomerase I inhibitors

Topoisomerase I (topo I) is an essential enzyme for vital cellular processes. Inhibition of topo I activities is lethal and leads to cell death, thus establishing topo I as a promising target for cancer treatment. Camptothecin, a natural alkaloid, inhibits topo I. Topotecan and irinotecan, synthetic derivatives of camptothecin, are the most potent anticancer drugs in clinical use. However, several limitations of camptothecins such as solubility, toxicity, stability, resistance and the required high drug dose have encouraged the development of non-camptothecin topo I inhibitors. Natural alkaloid benzo[c]phenanthridines and synthetic indenoisoquinolines have been extensively studied as alternatives to camptothecin. Interestingly, these non-camptothecin topo I inhibitors share a common 3-arylisoquinoline scaffold. This review will describe the development of novel indeno[1,2-c]isoquinolines, isoindolo[2,1-b]isoquinolines, 12-oxobenzo[c]phenanthridines and benz[b]oxepines with a 3-arylisoquinoline nucleus as topo I inhibitors.     

A method to accurately quantitate intensities of 32P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh

Quantitating relative ³²P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple ³²P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent ³²P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N²-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. V_max/K_m is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in V_max. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N²-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated from ³²P-signals or by other means (e.g., staining).     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Synthesis and biological evaluation of novel F-18 labeled pyrazolo[1,5-a]pyrimidine derivatives: potential PET imaging agents for tumor detection

Two novel pyrazolo[1,5-a]pyrimidine derivatives, 7-(2-[¹⁸F]fluoroethylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carbonitrile ([¹⁸F]FEMPPC, [¹⁸F]1) and N-(2-(3-cyano-5-methylpyrazolo[1,5-a]pyrimidin-7-ylamino)ethyl)-2-[¹⁸F]fluoro-4-nitrobenzamide ([¹⁸F]FCMPPN, [¹⁸F]2), have been designed and successively labeled with ¹⁸F by the nucleophilic substitution employing tosylate and nitryl as leaving groups, respectively. The radiochemical synthesis of both compounds was completed within 60 min with final high-performance liquid chromatography purification included. The corresponding radiochemical yields (without decay correction) were approximately 35% and 30%, respectively. Meanwhile, we compared the uptake characteristics of [¹⁸F]1 and [¹⁸F]2 with those of [¹⁸F]FDG and L-[¹⁸F]FET in S180 tumor cells. Furthermore, the tumor uptake of [¹⁸F]1 and [¹⁸F]2 was assessed in mice bearing S180 tumor and compared with [¹⁸F]FDG and L-[¹⁸F]FET in the same animal model. In vitro cell uptake studies showed [¹⁸F]1 had higher uptake than [¹⁸F]FDG, [¹⁸F]2 and L-[¹⁸F]FET over the 2 h period. In ex vivo biodistribution showed tumor/brain uptake ratios of [¹⁸F]2 were 12.35, 10.44, 8.69 and 5.13 at 15 min, 30 min, 60 min and 120 min post-injection, much higher than those of L-[¹⁸F]FET (2.43, 2.54, 2.93 and 2.95) and [¹⁸F]FDG (0.59, 0.61, 1.02 and 1.33) at the same time point. What’s more, the uptake of [¹⁸F]1 in tumor was 1.88, 4.37, 5.51, 2.95 and 2.88 at 5 min, 15 min, 30 min, 60 min and 120 min post-injection, respectively. There was a remarkable increasing trend before 30 min. The same trend was present for L-[¹⁸F]FET before 30 min and [¹⁸F]FDG before 60 min. Additionally, the tumor/brain uptake ratios of [¹⁸F]1 were superior to those of [¹⁸F]FDG at all the selected time points, the tumor/muscle and tumor/blood uptake ratios of [¹⁸F]1 at 30 min were higher than those of L-[¹⁸F]FET at the same time point. MicroPET image of [¹⁸F]1 administered into S180 tumor-bearing mouse acquired at 30 min post-injection illustrated that the uptake in S180 tumor was obvious. These results suggest that compound [¹⁸F]1 could be a new probe for PET tumor imaging.     

Reactions of the fac-[Re(CO)3] and [ReO] moieties with substituted benzothiazoles

The reaction of 2-(2-aminophenyl)benzothiazole (Habt) with [Re(CO)₅Br] led to the isolation of the rhenium(I) complex fac-[Re(Habt)(CO)₃Br] (1). With trans-[ReOCl₃(PPh₃)₂], the ligand Habt decomposed to form the oxofree rhenium(V) complex [Re(itp)₂Cl(PPh₃)] (2) (itp = 2-amidophenylthiolate). From the reaction of trans-[ReOBr₃(PPh₃)₂] with 2-(2-hydroxyphenyl)benzothiazole (Hhpd) the complex [Re^VOBr₂(hpd)(PPh₃)] (3) was obtained. Complexes 1-3 are stable and lipophilic. ¹H NMR and infrared assignments, as well as the X-ray crystal structures, of the complexes are reported.     

Dynamics of a benzo[a]pyrene-derived guanine DNA lesion in TGT and CGC sequence contexts: enhanced mobility in TGT explains conformational heterogeneity, flexible bending, and greater susceptibility to nucleotide excision repair

The nucleotide excision repair (NER) machinery excises a variety of bulky DNA lesions, but with varying efficiencies. The structural features of the DNA lesions that govern these differences are not well understood. An intriguing model system for studying structure-function relationships in NER is the major adduct derived from the reaction of the highly tumorigenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, with the exocyclic amino group of guanine ((+)-trans-anti-[BP]-N²-dG, or G*). The rates of incision of the stereochemically identical lesions catalyzed by the prokaryotic UvrABC system was shown to be greater by a factor of 2.3 ± 0.3 in the TG*T than in the CG*C sequence context [Biochemistry 46 (2007) 7006-7015]. Here we employ molecular dynamics simulations to elucidate the origin of the greater excision efficiency in the TG*T case and, more broadly, to delineate structural parameters that enhance NER. Our results show that the BP aromatic ring system is 5′-directed along the modified strand in the B-DNA minor groove in both sequence contexts. However, the TG*T modified duplex is much more dynamically flexible, featuring more perturbed and mobile Watson-Crick hydrogen bonding adjacent to the lesion, a greater impairment in stacking interactions, more dynamic local roll/bending, and more minor groove flexibility. These characteristics explain a number of experimental observations concerning the (+)-trans-anti-[BP]-N²-dG adduct in double-stranded DNA with the TG*T sequence context: its conformational heterogeneity in NMR solution studies, its highly flexible bend, and its lower thermal stability. By contrast, the CG*C modified duplex is characterized by a single BP conformation and a rigid bend. While current recognition models of bulky lesions by NER factors have stressed the importance of impaired Watson-Crick pairing/stacking and bending, our results highlight the likelihood of an important role for the local dynamics in the vicinity of the lesion.     

Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells

Reactive oxygen species (ROS) are responsible for lung damage during inhalation of cold air. However, the mechanism of the ROS production induced by cold stress in the lung is still unclear. In this work, we measured the changes of ROS and the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in A549 cell. We observed that cold stress (from 20 to 5 °C) exposure of A549 cell resulted in an increase of ROS and [Ca²⁺]_c, which was completely attenuated by removing Ca²⁺ from medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) agonist (allyl isothiocyanate, AITC) increased the production of ROS and the level of [Ca²⁺]_c in A549 cell. Moreover, HC-030031, a TRPA1 selective antagonist, significantly inhibited the enhanced ROS and [Ca²⁺]_c induced by AITC or cold stimulation, respectively. Taken together, these data demonstrated that TRPA1 activation played an important role in the enhanced production of ROS induced by cold stress in A549 cell.     

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N-dG) of the potent environmental carcinogen, benzo[a]pyrene

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question — what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? — has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N²-dG. In previous work, [+ta]-B[a]P-N²-dG was shown to be capable of inducing>95% G→T mutations in one sequence context (5′-T C), and 95% G→A mutations in another (5′-A A). This raises the question — how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N²-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G→T vs. G→A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N²-dG and AP-site mutagenesis, as well as the so-called “A-rule”.) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N²-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.     

Sup35 methionine oxidation is a trigger for de novo [PSI+] prion formation

ABSTRACT. The molecular basis by which fungal and mammalian prions arise spontaneously is poorly understood. A number of different environmental stress conditions are known to increase the frequency of yeast [PSI⁺] prion formation in agreement with the idea that conditions which cause protein misfolding may promote the conversion of normally soluble proteins to their amyloid forms. A recent study from our laboratory has shown that the de novo formation of the [PSI⁺] prion is significantly increased in yeast mutants lacking key antioxidants suggesting that endogenous reactive oxygen species are sufficient to promote prion formation. Our findings strongly implicate oxidative damage of Sup35 as an important trigger for the formation of the heritable [PSI⁺] prion in yeast. This review discusses the mechanisms by which the direct oxidation of Sup35 might lead to structural transitions favoring conversion to the transmissible amyloid-like form. This is analogous to various environmental factors which have been proposed to trigger misfolding of the mammalian prion protein (PrP^C) into the aggregated scrapie form (PrP^Sc).     

Efficient one-pot synthetic protocols for iminosugar-bearing imidazo[1,2-a]pyridines from carbohydrates

This letter describes two unprecedented one-pot high yielding synthetic approaches to imidazo[1,2-a]pyridine scaffolds from carbohydrates. The first approach involves microwave-assisted acid-catalyzed domino reactions of unprotected d-glucose/d-xylose with ammonium acetate and benzoin to afford polyhydroxy iminosugar-bearing tetrahydroimidazo[1,2-a]pyridines. In the second approach, polyhydroxy iminosugar-bearing tetrahydrobenzimidazo[1,2-a]pyridines were synthesized by using unprotected d-glucose/d-xylose and 1,2-diamines in the presence of 10 mol % of oxalic acid under solvent-free microwave irradiation conditions.