Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (T_m) and thermodynamic (−ΔG°) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the −ΔG° and T_m values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson–Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290–350 nm range is correlated linearly with −ΔG° and T_m, but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine–DNA adducts are recognized in nucleotide excision repair.     

Probing the sequence effects on NarI-induced -2 frameshift mutagenesis by dynamic 19F NMR, UV, and CD spectroscopy

The NarI recognition sequence (5'-G1G2CG3CN-3') is the most vulnerable hot spot for frameshift mutagenesis induced by the carcinogen 2-aminofluorene and its analogues in Escherichia coli. Lesioning of the guanine in the G3 position induces an especially high frequency of -2 deletion mutations; vulnerability to these mutations is modulated by the nature of the nucleotide in the N position (C approximately A > G > T). The objective of the present study was to probe the structural basis of this N-mediated influence on the propensity of the G3 lesion to form a slipped mutagenic intermediate (SMI) during translesion synthesis. We studied NarI-based fully paired [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNCGGCCGAG-3'), N = dC or dT] and -2 deletion [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNGCCGAG-3'), N = dC or dT] duplexes, in which G* was either AF [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene] or the 19F probe FAF [N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]. The latter sequences mimic the bulged SMI for -2 deletion mutations. Dynamic 19F NMR, circular dichroism, and UV melting results indicated that the NarI-dC/-2 deletion duplex adopts exclusively an intercalated conformer, whereas the NarI-dT/-2 deletion duplex exists as multiple conformers. The data support the presence of a putative equilibrium between a carcinogen-intercalated and a carcinogen-exposed SMI for the dT/-2 duplex. A similar dT-induced conformational heterogeneity was observed for the fully paired duplexes in which all three guanines were individually modified by AF or FAF. The frequency of the carcinogen stacked S-conformation was found to be highest (69-75%) at the G3 hot spot in NarI-dC duplexes. Taken together, our results support the hypothesis that the conformational stability of the SMI is a critical determinant for the efficacy of -2 frameshift mutagenesis in the NarI sequence. We also provide evidence for AF/FAF conformational compatibility in the NarI sequences.     

Influence of flanking sequence context on the conformational flexibility of aminofluorene-modified dG adduct in dA mismatch DNA duplexes

When positioned opposite to a dA in a DNA duplex, the prototype arylamine–DNA adduct [N-(2′-deoxyguanosin-yl)-7-fluoro-2-aminofluorene (FAF)] adopts the so-called ‘wedge’ (W) conformation, in which the carcinogen resides in the minor groove of the duplex. All 16 FAF-modified 12-mer NG*N/NAN dA mismatch duplexes (G* = FAF, N = G, A, C, T) exhibited strongly positive induced circular dichroism in the 290–360 nm range (ICD_{290–360 nm}), which supports the W conformation. The ICD_{290–360 nm} intensities were the greatest for duplexes with a 3′-flanking T. The AG*N duplex series showed little adduct-induced destabilization. An exception was the AG*T duplex, which displayed two well-resolved signals in the ¹⁹F NMR spectra. This was presumably due to a strong lesion-destabilizing effect of the 3′-T. The flanking T effect was substantiated further by findings with the TG*T duplex, which exhibited greater lesion flexibility and nucleotide excision repair recognition. Adduct conformational heterogeneity decreased in order of TG*T > AG*T > CG*T > AG*A > AG*G > AG*C. The dramatic flanking T effect on W-conformeric duplexes is consistent with the strong dependence of the ICD_290-360 on both temperature and salt concentration and could be extended to the arylamine food mutagens that are biologically relevant in humans.     

Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence

Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)～G(1)> G(2)). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G(2)～G(1)> G(3), a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.     

Covalent binding of a carcinogen as a probe for the dynamics of deoxyribonucleic acid

In order to determine the kinetic parameters of the binding to DNA of two closely related ultimate carcinogens, 2-(N-acetoxy-N-acetylamino)fluorene (N-Aco-AAF) and 2-(N-hydroxyamino)fluorene (N-OH-AF), three kinds of experiments were performed: measurement of the final amount of adduct (N-Aco-AAF and N-OH-AF), determination of the initial rate, and study of the reaction with deoxyguanosine (N-Aco-AAF only) at temperatures between 4 and 50 degrees C. The kinetic treatment of the chemical equations relies on two main assumptions: (i) binding of carcinogen to the C8 of guanine (G) could occur either with the classical B conformation or with a transient conformational state of the sugar--phosphate chain at the level of the guanine and denoted by G*; (ii) the equilibrium between G and G* is fast as compared to the chemical rate of carcinogen binding. These two assumptions have been verified by comparing experimental and calculated values of some of the data. From experimental data it is possible then to determine the characteristic independent parameters of the reaction: the constant K of the G in equilibrium G* and the enthalpy change delta H of the process, the rate constant k3 of the binding to the C8 of G, and the rate constant k1 of hydrolysis of the carcinogen with their corresponding activation enthalpies E3 and E1. Some essential results obtained are as follows: (a) The amount of G* that represents about 10% of the G at room temperature increases with temperature and is higher in denatured than in native DNA. (b) The values of delta H (approximately 9 kcal mol-1) and delta S (approximately 27 cal K-1 mol-1) of the G in equilibrium G* equilibrium are close to those associated with single base pair opening [Wartell, R.M., & Benight, A.S. (1982) Biopolymers 21, 2069]. (c) N-Aco-AAF reacts only with the G* conformation while N-OH-AF binds preferentially to the "classical" G (B conformation). Therefore, the electrophilic carcinogens behave as probes of the dynamic state of the DNA, but the rate of the G in equilibrium G* exchange is fast as compared to the binding rate of the carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Examination of the long-range effects of aminofluorene-induced conformational heterogeneity and its relevance to the mechanism of translesional DNA synthesis

Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the "B-DNA" major groove, S is "stacked" into the helix with base-displacement, and W is "wedged" into the minor groove. Here, we conducted (19)F NMR, CD, T(m), and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and T(m) results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.     

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling.

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.     

Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5'-d(…G*pT*…) and 5'-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.     

Sequence context- and temperature-dependent nucleotide excision repair of a benzo[a]pyrene diol epoxide-guanine DNA adduct catalyzed by thermophilic UvrABC proteins

The influence of DNA base sequence context on the removal of a bulky benzo[a]pyrene diol epoxide-guanine adduct, (+)-trans-B[a]P-N2-dG (G*), by UvrABC nuclease from the thermophilic organism Bacillus caldotenax was investigated. The lesion was flanked by either T or C in otherwise identical complementary 43-mer duplexes (TG*T or CG*C, respectively). It was reported earlier that in the CG*C context, a dominant minor groove adduct structure was observed by NMR methods with all Watson-Crick base pairs intact, and the duplex exhibited a rigid bend. In contrast, in the TG*T context, a highly flexible bend was observed, base pairing at G*, and two 5'-base pairs flanking the adduct were impaired, and multiple solvent-accessible adduct conformations were observed. The TG*T-43-mer duplexes are incised with consistently greater efficiency by UvrABC proteins from B. caldotenax by a factor of 2.3 +/- 0.3. The rates of incisions increase with increasing temperature and are characterized by linear Arrhenius plots with activation energies of 27.0 +/- 1.5 and 23.4 +/- 1.0 kcal/mol for CG*C and TG*T duplexes, respectively. These values reflect the thermophilic characteristics of the UVrABC nuclease complex and the contributions of the different DNA substrates to the overall activation energies. These effects are consistent with base sequence context-dependent differences in structural disorder engendered by a loss of local base stacking interactions and Watson-Crick base pairing in the immediate vicinity of the lesions in the TG*T duplexes. The local weakening of base pairing interactions constitutes a recognition element of the UvrABC nucleotide excision repair apparatus.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Molecular dynamics and free energy calculations of the B and Z forms of C8-arylguanine modified oligonucleotides

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8-tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6-31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5'-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.     

Site-specifically located 8-amino-2'-deoxyguanosine: thermodynamic stability and mutagenic properties in Escherichia coli

2-Nitropropane (2-NP), an important industrial solvent and a component of cigarette smoke, is mutagenic in bacteria and carcinogenic in rats. 8-Amino-2'-deoxyguanosine (8-amino-dG) is one of the types of DNA damage found in liver, the target organ in 2-NP-treated rats. To investigate the thermodynamic properties of 8-amino-dG opposite each of the four DNA bases, we have synthesized an 11mer, d(CCATCG*CTACC), in which G* represents the modified base. By annealing a complementary DNA strand to this modified 11mer, four sets of duplexes were generated each containing one of the four DNA bases opposite the lesion. Circular dichroism studies indicated that 8-amino-dG did not alter the global helical properties of natural right-handed B-DNA. The thermal stability of each duplex was examined by UV melting measurements and compared with its unmodified counterpart. For the unmodified 11mer, the relative stability of the complementary DNA bases opposite G was in the order C > T > G > A, as determined from their -DeltaG degrees values. The free energy change of each modified duplex was lower than its unmodified counterpart, except for the G*:G pair that exhibited a higher melting transition and a larger -DeltaG degrees than the G:G duplex. Nevertheless, the stability of the modified 11mer duplex also followed the order C > T > G > A when placed opposite 8-amino-dG. To explore if 8-amino-dG opposite another 8-amino-dG has any advantage in base pairing, a G*:G* duplex was evaluated, which showed that the stability of this duplex was similar to the G*:G duplex. Mutagenesis of 8-amino-dG in this sequence context was studied in Escherichia coli, which showed that the lesion is weakly mutagenic (mutation frequency approximately 10(-3)) but still can induce a variety of targeted and semi-targeted mutations.     

Molecular Dynamics and Free Energy Calculations of the B and Z Forms of C8-Arylguanine Modified Oligonucleotides

Abstract

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8- tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6–31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5′-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.     

L-nucleotides and 8-methylguanine of d(C1m8G2C3G4C5LG6LC7G8C9G10)2 act cooperatively to promote a left-handed helix under physiological salt conditions

The structure and thermal stability of a hetero chiral decaoligodeoxyribonucleotide duplex d(C1m8 G2C3G4C5LG6LC7G8C9G10)d(C11m8G12C13G14C15LG16LC17G18C19G20) (O1) with two contiguous pairs of enantiomeric 2'-deoxy-L-ribonucleotides (C5LG6L/C15LG16L) at its centre and an 8-methylguanine at position 2/12 was analysed by circular dichroism, NMR and molecular modelling. O1 resolves in a left-handed helical structure already at low salt concentration (0.1 M NaCl). The central L2-sugar portion assumes a B* left-handed conformation (mirror-image of right-handed B-DNA) while its flanking D4-sugar portions adopt the known Z left-handed conformation. The resulting Z4-B2*-Z4 structure (left-handed helix) is the reverse of that of B4-Z2*-B4 (right-handed helix) displayed by the nearly related decaoligodeoxyribonucleotide d(mC1G2mC3G4C5L G6LmC7G8mC9G10)2, at the same low salt concentration (0.1 M NaCl). In the same experimental conditions, d(C1m8G2C3G4C5G6C7G8C9G10)2 (O2), the stereoregular version of O1, resolves into a right-handed B-DNA helix. Thus, both the 8-methylguanine and the enantiomeric step CLpGL at the centre of the molecule are needed to induce left-handed helicity. Remarkably, in the various heterochiral decaoligodeoxyribonucleotides so far analysed by us, when the central CLpGL adopts the B* (respectively Z*) conformation, then the adjacent steps automatically resolves in the Z (respectively B) conformation. This allows a good optimisation of the base-base stackings and base-sugar van der Waals interactions at the ZB*/B*Z (respectively BZ*/Z*B) junctions so that the Z4-B2*-Z4 (respectively B4-Z2*-B4) helix displays a Tm (approximately 65 degrees C) that is only 5 degrees C lower than the one of its homochiral counterpart. Here we anticipate that a large variety of DNA helices can be generated at low salt concentration by manipulating internal factors such as sugar configuration, duplex length, nucleotide composition and base methylation. These helices can constitute powerful tools for structural and biological investigations, especially as they can be used in physiological conditions.     

Energetic and conformational contributions to the stability of Okazaki fragments

A combination of spectroscopic and calorimetric techniques was used to determine complete thermodynamic profiles accompanying the folding of a model Okazaki fragment with sequence 5'-r(gagga)d(ATCTTTG)-3'/5'-d(CAAAGATTCCTC)-3' and control DNA (with and without thymidine substitutions for uridine), RNA, and hybrid duplexes. Circular dichroism spectroscopy indicated that all DNA duplexes are in the B conformation, the RNA and hybrid duplexes are in the A conformation, and the Okazaki fragment exhibits a spectrum between the A and B conformations. Ultraviolet and differential scanning calorimetry melting experiments reveal that all duplexes unfold in two-state transitions with thermal stabilities that follow the order RNA > OKA > DNA (with thymidines) > hybrids > DNA (with uridines). The dependence of the transition temperature on salt concentration yielded counterion releases in the following order: DNA (with thymidines) > RNA > DNA (with uridines) > OKA > hybrids. Thus, Okazaki fragments have a conformation and charge density between those of its components DNA and hybrid segments. However, the presence of the RNA-DNA/DNA junction confers on them higher stabilities than their component hybrid and DNA segments. The binding of intercalators to an Okazaki hairpin of sequence 5'-r(gc)d(GCU5GCGC)-3' and to its control DNA hairpin has also been studied. The results show that the binding of intercalators to Okazaki fragments is accompanied with higher heats and lower binding affinities, compared with DNA duplexes. This suggests that the presence of an RNA/DNA junction yields a larger surface contact to interact with the phenanthroline ring of the intercalators, which may lead to a larger disruption of the flexible flanking bases of the junction. The overall results suggest that the presence of this junction stabilizes Okazaki fragments and provides a structural feature that can be exploited in the design of drugs to specifically target these molecules.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.     

DFT study of alpha- and beta-D-mannopyranose at the B3LYP/6-311++G** level

Thirty-five conformations of alpha- and beta-d-mannopyranose, the C-2 substituted epimer of glucopyranose, were geometry optimized using the density functional (B3LYP), and basis set (6-311++G**). Full geometry optimization was performed on the hydroxymethyl rotamers (gg/gt/tg) and an analytical hessian program was used to calculate the harmonic vibrational frequencies, zero point energy, enthalpy, and entropy. The lowest energy conformation investigated is the beta-tg in the (4)C(1) chair conformation. The in vacuo calculations showed little energetic preference for either the alpha or beta anomer for mannopyranose in the (4)C(1) chair conformation. Results are compared to similar glucopyranose calculations in vacuo where the alpha anomer is approximately 1kcal/mol lower in electronic energy than the beta anomer. In the case of the generally higher energy (1)C(4) chair conformations, one low-energy, low-entropy beta-gg-(1)C(4) chair conformation was identified that is within approximately 1.4kcal/mol of the lowest energy (4)C(1) conformation of mannopyranose. Other (1)C(4) chair conformations in our investigation are approximately 2.9-7.9kcal/mol higher in overall energy. Many of the (3,O)B, B(3,O), (1,4)B, and B(1,4) boat forms passed through transitions without barriers to (1)S(3), (5)S(1), (1)S(5) skew forms with energies between approximately 3.6 and 8.9kcal/mol higher in energy than the lowest energy conformation of mannopyranose. Boat forms were found that remained stable upon gradient optimization. As with glucopyranose, the orientation and interaction of the hydroxy groups make a significant contribution to the conformation/energy relationship in vacuo.     

The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions

Nucleotide excision repair (NER) is a vital cellular defense system against carcinogen-DNA adducts, which, if not repaired, can initiate cancer development. The structural features of bulky DNA lesions that account for differences in NER efficiencies in mammalian cells are not well understood. In vivo, the predominant DNA adduct derived from metabolically activated benzo[a]pyrene (BP), a prominent environmental carcinogen, is the 10S (+)-trans-anti-[BP]-N²-dG adduct (G*), which resides in the B-DNA minor groove 5′-oriented along the modified strand. We have compared the structural distortions in double-stranded DNA, imposed by this adduct, in the different sequence contexts 5′-…CGG*C…, 5′-…CG*GC…, 5′-…CIG*C… (I is 2′-deoxyinosine), and 5′-…CG*C…. On the basis of electrophoretic mobilities, all duplexes manifest moderate bends, except the 5′-…CGG*C…duplex, which exhibits an anomalous, slow mobility attributed to a pronounced flexible kink at the site of the lesion. This kink, resulting from steric hindrance between the 5′-flanking guanine amino group and the BP aromatic rings, both positioned in the minor groove, is abolished in the 5′-…CIG*C…duplex (the 2′-deoxyinosine group, I, lacks this amino group). In contrast, the sequence-isomeric 5′-…CG*GC…duplex exhibits only a moderate bend, but displays a remarkably increased opening rate at the 5′-flanking base pair of G*, indicating a significant destabilization of Watson-Crick hydrogen bonding. The NER dual incision product yields were compared for these different sequences embedded in otherwise identical 135-mer duplexes in cell-free human HeLa extracts. The yields of excision products varied by a factor of as much as ∼ 4 in the order 5′-...CG*GC…> 5′...CGG*C…≥ 5′...CIG*C…≥ 5′-…CG*C…. Overall, destabilized Watson-Crick hydrogen bonding, manifested in the 5′-...CG*GC...duplex, elicits the most significant NER response, while the flexible kink displayed in the sequence-isomeric 5′-...CGG*C...duplex represents a less significant signal in this series of substrates. These results demonstrate that the identical lesion can be repaired with markedly variable efficiency in different local sequence contexts that differentially alter the structural features of the DNA duplex around the lesion site.