DNA Deoxyribose Research Articles

Electric Field Promoted Click Surface-Enhanced Raman Spectroscopy for Rapid and Specific Detection of DNA 2-Deoxyribose 5'-Aldehyde Oxidation Products in Plasma.

Rapid identification of DNA oxidative damage sites is of great significance for disease diagnosis. In this work, electric field-regulated click reaction surface-enhanced Raman spectroscopy (e-Click-SERS) was developed aiming at the rapid and specific analysis of furfural, the biomarker of oxidative damage to the 5-carbon site of DNA deoxyribose. In e-Click-SERS, cysteamine-modified porous Ag filaments (cys@p-Ag) were prepared and used as electrodes, amine-aldehyde click reaction sites, and SERS substrates. Cysteamine was controlled as an "end-on" conformation by setting the voltage of cys@p-Ag at -0.1 V, which ensures its activity in participating in the amine-aldehyde click reaction during the detection of furfural. Benefiting from this, the proposed e-Click-SERS method was found to be sensitive, rapid-responding, and interference-resistant in analyzing furfural from plasma. The method detection limits of furfural were 5 ng mL-1 in plasma, and the whole "extraction and detection" procedure was completed within 30 min with satisfactory recovery. Interference from 13 kinds of common plasma metabolites was investigated and found to not interfere with the analysis, according to the exclusive adaptation of the amine-aldehyde click reaction. Notably, the e-Click-SERS technique allows in situ analysis of biological samples, which offers great potential to be a point-of-care testing tool for detecting DNA oxidative damage.

Monte Carlo simulations of electron interactions with the DNA molecule: A complete set of physics models for Geant4-DNA simulation toolkit

In this study we are introducing an update of the Geant4-DNA physics constructor “option 6” including electron interactions with all constituents of the DNA molecule in addition to those already publicly available for liquid water. The new implementation is based on the interaction cross sections of electrons with the four DNA nucleobases, deoxyribose and phosphoric acid for elastic scattering, electronic excitation and ionisation in the 11 eV – 1 MeV energy range. An additional sampling method to estimate the transferred secondary electron energy produced by ionisation is also introduced and can be optionally activated instead of the classical interpolation method based on the differential cross section tables, thus eliminating the need to upload large data files. The implementation in Geant4-DNA was verified by calculating range and electronic stopping power in the various materials. Good agreement is observed with the data available in the literature, and calculations with the interpolation method and the sampling method showed less than 4% difference. No differences were observed in terms of computational cost.

NMR solution structure of tricyclo-DNA containing duplexes: insight into enhanced thermal stability and nuclease resistance.

Tc-DNA is a conformationally constrained oligonucleotide analogue which shows significant increase in thermal stability when hybridized with RNA, DNA or tc-DNA. Remarkably, recent studies revealed that tc-DNA antisense oligonucleotides (AO) hold great promise for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy. To date, no high-resolution structural data is available for fully modified tc-DNA duplexes and little is known about the origins of their enhanced thermal stability. Here, we report the structures of a fully modified tc-DNA oligonucleotide paired with either complementary RNA, DNA or tc-DNA. All three investigated duplexes maintain a right-handed helical structure with Watson-Crick base pairing and overall geometry intermediate between A- and B-type, but closer to A-type structures. All sugars of the tc-DNA and RNA residues adopt a North conformation whereas the DNA deoxyribose are found in a South-East-North conformation equilibrium. The conformation of the tc-DNA strand in the three determined structures is nearly identical and despite the different nature and local geometry of the complementary strand, the overall structures of the examined duplexes are very similar suggesting that the tc-DNA strand dominates the duplex structure.

The Mechanism of Action of (-)-Lomaiviticin A.

(-)-Lomaiviticin A (4) is a complex C2-symmetric bacterial metabolite that contains two diazofluorene functional groups. The diazofluorene consists of naphthoquinone, cyclopentadiene, and diazo substituents fused through a σ- and π-bonding network. Additionally, (-)-lomaiviticin A (4) is a potent cytotoxin, with half-maximal inhibitory potency (IC50) values in the low nanomolar range against many cancer cell lines. Because of limitations in supply, its mechanism of action had remained a "black box" since its isolation in the early 2000s. In this Account, I describe how studies directed toward the total synthesis of (-)-lomaiviticin A (4) provided a platform to elucidate the emergent properties of this metabolite and thereby connect chemical reactivity with cellular phenotype. We first developed a convergent strategy to prepare the diazofluorene (9 + 10 → 13). We then adapted this chemistry to the synthesis of lomaiviticin aglycon (21/22) and the natural monomeric diazofluorene (-)-kinamycin F (3). The key step in the lomaiviticin aglycon (21/22) synthesis involved the stereoselective oxidative coupling of two monomeric diazofluorenes (2 × 18 → 20) to establish the cojoining carbon-carbon bond of the target. As the absolute stereochemistry of the aglycon and carbohydrate residues of (-)-lomaiviticin A (4) were unknown, we developed a semisynthetic route to the metabolite that proceeds in one step and 42% yield by diazo transfer to the more abundant isolate (-)-lomaiviticin C (6). This allowed us to complete the stereochemical assignment of (-)-lomaiviticin A (4) and provided a renewable source of material. Using this material, we established that the remarkable cytotoxic effects of (-)-lomaiviticin A (4) derive from the induction of highly toxic double-strand breaks (DSBs) in DNA. At the molecular level, 1,7-nucleophilic additions to each electrophilic diazofluorene trigger homolytic decomposition pathways that produce sp2 radicals at the carbon atoms of each diazo group. These radicals abstract hydrogen atoms from the deoxyribose of DNA, a process known to initiate strand cleavage. NMR spectroscopy and molecular mechanics simulations were used to elucidate the mode of DNA binding. These studies showed that both diazofluorenes of (-)-lomaiviticin A (4) penetrate into the duplex. This mode of non-covalent binding places each diazo carbon atom in close proximity to each DNA strand. Throughout these studies, isolates containing one diazofluorene, such as (-)-lomaiviticin C (6) and (-)-kinamycin C (2), were used as controls. Consistent with our mechanistic model, these compounds do not induce DSBs in DNA and are several orders of magnitude less potent. Reactivity studies suggest that (-)-lomaiviticin A (4) is more electrophilic than simple monomeric diazofluorenes. We attribute this to through-space delocalization of the developing negative charge in the transition state for 1,7-addition. Consistent with this mechanism of action, (-)-lomaiviticin A (4) displays selective low-picomolar potencies toward DNA DSB repair-deficient cell types. The emergent properties of (-)-lomaiviticin A (4) derive from the specific arrangement of diazo, naphthoquinone, cyclopentadiene, and ketone functional groups. These functional groups work together to yield, essentially, a masked vinyl radical that can be exposed under biological conditions. Furthermore, the rotational symmetry of the metabolite, deriving from dimerization, allows it to interact with the antiparallel symmetry of DNA and affect cleavage of the duplex.

Copper complexes as artificial DNA metallonucleases: From Sigman’s reagent to next generation anti-cancer agent?

Abstract Platinum drugs continue to dominate medical oncology treatments but they have shortcomings associated with their use including dose-limiting toxic side effects and acquired or intrinsic drug resistance. Copper complexes have been extensively investigated as an alternative class of cancer therapeutic. In contrast to Pt drugs which covalently bind DNA nucleobases, Cu derivatives predominantly form non-covalent interactions with DNA – either via intercalation, electrostatic forces of attraction and/or major or minor groove binding. Sigman et al. pioneered research in the late 1970s into Cu complexes as potential chemical nuclease agents. Copper complexes have since been shown to induce DNA cleavage via nucleobase oxidation, hydrolysis of DNA phosphate esters and/or deoxyribose sugar oxidation. The scope of this review covers the period 2014 – present, with a distinct focus on Cu(II) systems which have been investigated as chemical nuclease agents. Specifically we describe how Cu complexes exert their chemical nuclease activity prior to presenting an overview of Cu complexes incorporating Schiff bases, amino acids, peptides, azoles, terpyridines or polypyridyls as ligands as well as including a section on dinuclear Cu complexes and Cu complexes incorporating natural products or bioactive ligands.

DNA with Ionic, Atomic, and Clustered Silver: An XPS Study

The rapidly developing field of bionanotechnology requires detailed knowledge of the mechanisms of interaction between inorganic matter and biomolecules. Under conditions different from those in an aqueous solution, however, the chemistry of these systems is elusive and may differ dramatically from their interactions in vitro and in vivo. Here, we report for the first time a photoemission study of a metal silver-DNA interface, formed in vacuo, in comparison with DNA-Ag+ and fluorescent DNA-Ag complexes formed in solution. The high-resolution photoelectron spectra reveal that in vacuo silver atoms interact mainly with oxygen atoms of the phosphodiester bond and deoxyribose in DNA, in contrast to the behavior of silver ions, which interact preferentially with the nitrogen atoms of the bases. This offers new insight into the mechanism of DNA metallization, which is of importance in creating metal-bio interfaces for nanotechnology applications.

Enzyme mechanism-based, oxidative DNA–protein cross-links formed with DNA polymerase β in vivo

Free radical attack on the C1' position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA-protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-DPC under 1% O2 than under 21% O2) and even more robustly with the "chemical nuclease" 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-DPC had an unexpectedly short half-life (∼ 30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them.

Landscape of π–π and sugar–π contacts in DNA–protein interactions

There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA–protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π–π (according to biological edge) or deoxyribose sugar–π (according to sugar edge). Overall, 54% of contacts are nucleobase π–π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar–π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase–cyclic amino acid contacts, sugar–π interactions are more common than nucleobase π–π contacts for the acyclic series (61% of contacts). Similar to DNA–protein π–π interactions, sugar–π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA–protein π-interactions stabilize biological systems, by up to approximately −40 kJ mol−1 for neutral nucleobase or sugar–amino acid interactions, but up to approximately −95 kJ mol−1 for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.

Structure and Mechanism of DNA Polymerase β

DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.

DNA-protein π-interactions in nature: abundance, structure, composition and strength of contacts between aromatic amino acids and DNA nucleobases or deoxyribose sugar.

Four hundred twenty-eight high-resolution DNA–protein complexes were chosen for a bioinformatics study. Although 164 crystal structures (38% of those searched) contained no interactions, 574 discrete π–contacts between the aromatic amino acids and the DNA nucleobases or deoxyribose were identified using strict criteria, including visual inspection. The abundance and structure of the interactions were determined by unequivocally classifying the contacts as either π–π stacking, π–π T-shaped or sugar–π contacts. Three hundred forty-four nucleobase–amino acid π–π contacts (60% of all interactions identified) were identified in 175 of the crystal structures searched. Unprecedented in the literature, 230 DNA–protein sugar–π contacts (40% of all interactions identified) were identified in 137 crystal structures, which involve C–H···π and/or lone–pair···π interactions, contain any amino acid and can be classified according to sugar atoms involved. Both π–π and sugar–π interactions display a range of relative monomer orientations and therefore interaction energies (up to –50 (–70) kJ mol−1 for neutral (charged) interactions as determined using quantum chemical calculations). In general, DNA–protein π-interactions are more prevalent than perhaps currently accepted and the role of such interactions in many biological processes may yet to be uncovered.

Strain dependent UV degradation of Escherichia coli DNA monitored by Fourier transform infrared spectroscopy

In this work we present a method for detection of DNA isolated from nonpathogenic Escherichia coli strains, respectively. Untreated and UV irradiated bacterial DNAs were analyzed by FT-IR spectroscopy, to investigate their screening characteristic features and their structural radiotolerance at 253.7nm.FT-IR spectra, providing a high molecular structural information, have been analyzed in the wavenumber range 800–1800cm−1. FT-IR signatures, spectroscopic band assignments and structural interpretations of these DNAs are reported.Also, UV damage at the DNA molecular level is of interest. Strain dependent UV degradation of DNA from E. coli has been observed. Particularly, alterations in nucleic acid bases, base pairing and base stacking have been found. Also changes in the DNA conformation and deoxyribose were detected.Based on this work, specific E. coli DNA-ligand interactions, drug development and vaccine design for a better understanding of the infection mechanism caused by an interference between pathogenic and nonpathogenic bacteria and for a better control of disease, respectively, might be further investigated using Fourier transform infrared spectroscopy. Besides, understanding the pathways for UV damaged DNA response, like nucleic acids repair mechanisms is appreciated.

AbstractsAstrobiology Graduate Conference (AbGradCon) 2011HOSTED BYMontana State UniversityBozeman, Montana • June 5–8, 2011

AbstractsAstrobiology Graduate Conference (AbGradCon) 2011HOSTED BYMontana State UniversityBozeman, Montana • June 5–8, 2011

Setting Anchor in the Minor Groove: in Silico Investigation into Formamido N-Methylpyrrole and N-Methylimidazole Polyamides Bound by Cognate DNA Sequences

Tricyclic N-methylpyrrole (Py) and N-methylimidazole (Im) containing polyamide monocations are known to bind as stacked dimers within the minor groove of DNA, and those with N-terminal formamido (f) substituents bind in a staggered configuration with high specificity over a range of affinities. Although binding constants have been reported, there is not a clear understanding of why such constants vary significantly for polyamide dimers and their respective cognate DNA sequences. By employing computational tools, the following homodimer complexes have been addressed in this study: f-PyPyIm in complex with 5'-d(GAACTAGTTC)-3', f-ImPyPy in complex with 5'-d(GAATGCATTC)-3', and f-ImPyIm in complex with 5'-d(GAACGCGTTC)-3'. These complexes were selected based on their 10- to 100-fold differences in binding constants. From this study, it was possible to determine how polyamides anchor themselves within the minor groove of specific DNA sequences. This is done through several interactions that provide stability for specific recognition: (i) Py groups secure themselves between DNA base pairs, (ii) lone-pair-Π interactions are formed between DNA deoxyribose O4' and Im groups nearest f, (iii) minor groove bases hydrogen bond to Im groups and amides of the polyamide backbone, (iv) f substituents rotate without leaving the minor groove of DNA and with this rotation form specific hydrogen bonds with electron-rich sites on the floor of the minor groove, and (v) flexible charged N,N-dimethylaminoalkyl substituents reside favorably in the minor groove of DNA. Results displayed the greatest amount of interactions and stability for dimer f-ImPyIm in complex with 5'-d(GAACGCGTTC)-3' and the least amount in dimer f-PyPyIm in complex with 5'-d(GAACTAGTTC)-3'. Hence, for cognate DNA sequences, the relative binding strength of compounds was determined as f-ImPyIm > f-ImPyPy > f-PyPyIm. This force-field-based computational study is in agreement with experimental results and provides a molecular rational for the binding constant values.

Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

BackgroundDNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB).Principal FindingsHere, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ.ConclusionsThe expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.

Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Mammalian DNA polymerase β is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymeraseβ, TcPolβ and TcPolβPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolβPAK, in comparison to TcPolβ, conducted DNA synthesis over a much broader pH range. TcPolβ was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolβ were not observed for TcPolβPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolβ, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

On the Chemical Yield of Base Lesions, Strand Breaks, and Clustered Damage Generated in Plasmid DNA by the Direct Effect of X Rays

The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.

N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N(6)-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3'-formylphosphate residues arising from 5'-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N(6)-amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N(6)-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04-0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose-response relationship for the formation of N(6)-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N(6)-formylation of the linker histone H1. The N(6)-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N(6)-acetylation recognized as an important determinant of gene expression in mammalian cells. The N(6)-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

Formation of 1,4-Dioxo-2-butene-Derived Adducts of 2‘-Deoxyadenosine and 2‘-Deoxycytidine in Oxidized DNA

Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M(1)dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the beta-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5'-oxidation of deoxyribose in DNA, and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5'-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts.

Dissociative electron attachment to furan, tetrahydrofuran, and fructose

We study dissociative electron attachment to furan (FN) (C(4)H(4)O), tetrahydrofuran (THF) (C(4)H(8)O), and fructose (FRU) (C(6)H(12)O(6)) using crossed electron/molecular beams experiments with mass spectrometric detection of the anions. We find that FN and THF are weak electron scavengers and subjected to dissociative electron attachment essentially in the energy range above 5.5 eV via core excited resonances. In striking contrast to that, FRU is very sensitive towards low energy electrons generating a variety of fragment ions via a pronounced low energy feature close to 0 eV. These reactions are associated with the degradation of the ring structure and demonstrate that THF cannot be used as surrogate to model deoxyribose in DNA with respect to the attack of electrons at subexcitation energies (<3 eV). The results support the picture that in DNA the sugar moiety itself is an active part in the initial molecular processes leading to single strand breaks.

Structure and Mechanism of DNA Polymerase β

DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.