The influence of nearest neighbours on the efficiency of coaxial stacking at contiguous stacking hybridization of oligodeoxyribonucleotides

Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV-melting technique. It was shown that the free-energy of the coaxial stacking interaction (deltaG degrees ST at 37 degrees C, 1 M NaCl, pH 7.4) at the complementary interface XA*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA*pTPu/PyATPu interface. A 5'-phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A*T/AT coaxial stacking varied from (- 0.97 kcal/mol) for unphosphorylated TA*TA/TATA interface to three-fold higher value (- 2.78 kcal/mol) for GA*pTT/AATC interface.     

Thermodynamic analysis of stacking hybridization of oligonucleotides with DNA template.

Contiguous stacking hybridization of oligodeoxyribonucleotides with DNA as template was investigated using three types of complexes: oligonucleotide contiguously stacked with the stem of the preformed minihairpin (complexes I), oligonucleotide tandems containing two (complexes II) or three (complexes III) short oligomers with a common DNA template. Enthalpy Delta H degrees and entropy Delta S degrees of the coaxial stacking of adjacent duplexes were determined for GC/G*pC, GT/A*pC, AC/G*pT, AT/A*pT, CT/A*pG, AG/C*pT, AA/T*pT and TT/A*pA nicked (*) dinucleotide base pairs. The maximal efficiency of co-operative interaction was found for the GC/G*pC interface (Delta G degrees(NN/N*pN)=-2.7 kcal/mol) and the minimal one for the AA/T*pT interface (Delta G degrees(NN/N*pN)=-1.2 kcal/mol) at 37 degrees C. As a whole, the efficiency of the base pairs interaction Delta G degrees(NN/N*pN) in the nick is not lower than that within the intact DNA helix (Delta G degrees(NN/NN)).These observed Delta G degrees(NN/N*pN) values are proposed may include the effect of the partial removal of fraying at the adjacent helix ends additionally to the effect of the direct stacking of the terminal base pairs in the duplex junction (Delta G degrees(NN/NN). The thermodynamic parameters have been found to describe adequately the formation of all tandem complexes of the II and III types with oligonucleotides of various length and hybridization properties. The performed thermodynamic analysis reveals features of stacking oligonucleotide hybridization which allow one to predict the temperature dependence of association of oligonucleotides and the DNA template within tandem complexes as well as to determine optimal concentration for formation of these complexes characterized by high co-operativity level.     

Mutational analysis of the RNA pseudoknot involved in efficient ribosomal frameshifting in simian retrovirus-1.

Mutational effects on frameshifting efficiency of the RNA pseudoknot involved in ribosomal frameshifting in simian retrovirus-1 (SRV-1) have been investigated. The primary sequence and the proposed secondary structure of the SRV-1 pseudoknot are similar to those of other efficient frameshifting pseudoknots in mouse mammary tumor virus (MMTV) and feline immunodeficiency virus (FIV), where an unpaired adenine nucleotide intercalates between stem 1 and stem 2. In SRV-1 pseudoknot, the adenine nucleotide in between stem 1 and stem 2 has a potential to form an A*U base pair with the last uridine nucleotide in the loop 2, resulting in a continuous A-form helix with coaxially stacked stem 1 and stem 2. To test whether this A*U base pairing and coaxial stacking of stem 1 and stem 2 is absolutely required for efficient frameshifting in SRV-1, a series of mutants changing this potential A.U base pair to either G.C base pair or A.A, A.G, A.C, G.A, G.G mismatch is generated, and their frameshifting efficiencies are investigated in vitro using rabbit reticulocyte lysate translation assay. The frameshifting abilities of these mutant pseudoknots are similar to that of the wild-type pseudoknot, suggesting that the A*U base pair in between stem 1 and stem 2 is not necessary to promote efficient frameshifting in SRV-1. These results reveal that coaxial stacking of stem 1 and stem 2 with a Watson-Crick A.U base pair in between two stems is not a required structural feature of the pseudoknot for promoting efficient frameshifting in SRV-1. Our mutational data suggest that SRV-1 pseudoknot adopts similar structural features common to other efficient frameshifting pseudoknots as observed in MMTV and FIV.     

The Influence of Nearest Neighbours on the Efficiency of Coaxial Stacking at Contiguous Stacking Hybridization of Oligodeoxyribonucleotides

Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV‐melting technique. It was shown that the free‐energy of the coaxial stacking interaction (ΔG°_ST at 37°C, 1 M NaCl, pH 7.4) at the complementary interface XA^*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA^*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA^*pTPu/PyATPu interface. A 5′‐phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A^*T/AT coaxial stacking varied from (? 0.97 kcal/mol) for unphosphorylated TA^*TA/TATA interface to three‐fold higher value (? 2.78 kcal/mol) for GA^*pTT/AATC interface.     

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.     

Predicting helical coaxial stacking in RNA multibranch loops

The hypothesis that RNA coaxial stacking can be predicted by free energy minimization using nearest-neighbor parameters is tested. The results show 58.2% positive predictive value (PPV) and 65.7% sensitivity for accuracy of the lowest free energy configuration compared with crystal structures. The probability of each stacking configuration can be predicted using a partition function calculation. Based on the dependence of accuracy on the calculated probability of the stacks, a probability threshold of 0.7 was chosen for predicting coaxial stacks. When scoring these likely stacks, the PPV was 66.7% at a sensitivity of 51.9%. It is observed that the coaxial stacks of helices that are not separated by unpaired nucleotides can be predicted with a significantly higher accuracy (74.0% PPV, 66.1% sensitivity) than the coaxial stacks mediated by noncanonical base pairs (55.9% PPV, 36.5% sensitivity). It is also shown that the prediction accuracy does not show any obvious trend with multibranch loop complexity as measured by three different parameters.     

Parallel multiplex thermodynamic analysis of coaxial base stacking in DNA duplexes by oligodeoxyribonucleotide microchips

Parallel thermodynamic analysis of the coaxial stacking effect of two bases localized in one strand of DNA duplexes has been performed. Oligonucleotides were immobilized in an array of three-dimensional polyacrylamide gel pads of microchips (MAGIChips‘). The stacking effect was studied for all combinations of two bases and assessed by measuring the increase in melting temperature and in the free energy of duplexes formed by 5mers stacked to microchip-immobilized 10mers. For any given interface, the effect was studied for perfectly paired bases, as well as terminal mismatches, single base overlaps, single and double gaps, and modified terminal bases. Thermodynamic parameters of contiguous stacking determined by using microchips closely correlated with data obtained in solution. The extension of immobilized oligonucleotides with 5,6-dihydroxyuridine, a urea derivative of deoxyribose, or by phosphate, decreased the stacking effect moderately, while extension with FITC or Texas Red virtually eliminated stacking. The extension of the immobilized oligonucleotides with either acridine or 5-nitroindole increased stacking to mispaired bases and in some GC-rich interfaces. The measurements of stacking parameters were performed in different melting buffers. Although melting temperatures of AT- and GC-rich oligonucleotides in 5 M tetramethylammonium chloride were equalized, the energy of stacking interaction was significantly diminished.     

利用SNP标记及配合力划分超甜玉米自交系的杂种优势群

划分超甜玉米自交系的杂种优势群,筛选配合力高的甜玉米自交系,构建新的杂种优势更强的甜玉米群体,为优良甜玉米的选育提供依据.本研究选用23个自育超甜玉米自交系,采用NC Ⅱ设计得到60(3×20)个组合,研究它们的产量配合力效应并结合56K SNP标记将供试材料进行杂种优势群的划分.结果表明23个自交系的平均杂合率为2....     

Structural and electronic properties of 4H-cyclopenta[2,1-b,3;4-b′]dithiophene S-oxide (BTO) derivatives with an S, S=O, O, SiH2, or BH2 bridge: semi-empirical and DFT study

In this paper, we theoretically studied the geometries, stabilities, and the electronic and thermodynamic properties of 4H-cyclopenta[2,1-b,3;4-b']dithiopene S-oxide derivatives (BTO-X, with X = BH(2), SiH(2), S, S=O, or O) using semi-empirical methods, ab initio methods, and density functional theory. The geometries and thermodynamic parameters calculated by PM3 were in good agreement with those calculated with B3LYP/6-31 G*. The band gap calculated using B3LYP/6-31 G* ranged from 3.94 eV (BTO-O) to 3.16 eV (BTO-B). The absorption λ(max) calculated using B3LYP/6-31 G* was shifted to longer wavelengths when X = BH(2), SiH(2), or S=O (due to their electron-withdrawing effects) and to shorter wavelengths for BTO-S and BTO-O as compared to the λ(max) for the thiophene S-oxide (2TO) dimer. The changes in ΔH°, ΔS°, and ΔG° calculated using both semi-empirical and DFT methods were quite similar.     

Functional evaluation of GJB2 variants in nonsyndromic hearing loss

Mutations in the gap junction β2 (GJB2) gene, encoding the connexin26 (CX26) protein, are the most common cause of non-syndromic hearing loss (HL) in many populations. In the East Asian population, two variants, p.V27I (c.79G>A) and p.E114G (c.341G>A), are considered benign polymorphisms since these variants have been identified in both HL patients and normal hearing controls. However, some studies have postulated that homozygotes carrying both p.V27I and p.E114G variants could cause HL. To elucidate possible roles of these variants, we used in vitro approaches to directly assess the pathogenicity of four haplotypes generated by the two polymorphisms: VE (wild type), I*E (p.V27I variant only), VG* (p.E114G variant only), I*G* (both variants). In biochemical coupling assays, the gap junctions (GJs) composed of VG* and I*G* types displayed defective channel activities compared with those of VE wild types or I*E types, which showed normal channel activities. Interestingly, the defect in hemichannel activity was a bit less severe in I*G* type than VG* type, suggesting that I* variant (p.V27I) may compensate for the deleterious effect of G* variant (p.E114G) in hemichannel activities. Our population studies using 412 Korean individuals showed that I*G* type was detected at around 20% in both HL patients and normal controls, suggesting that I*G* type may not be a pathogenic polymorphism. In contrast, VG* type was very rare (3/824) and detected only in HL patients, suggesting that VG* homozygotes (VG*/VG*) or compound heterozygotes carrying VG* type with other mutations may cause HL.     

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Analysis of melting transitions of the DNA hairpins formed from the oligomer sequences d[GGATAC(X)4GTATCC] (X = A, T, G, C)

Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3 degrees C for the T4 loop hairpin to 55.3 degrees C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop formation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions. Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop.(ABSTRACT TRUNCATED AT 400 WORDS)     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe- to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (DeltaG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 degrees C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA-] radical pair to be -0.43+/-0.025 eV, at 10 mus after the photon absorption event for PSII in its S(3)-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA-] is calculated to be -0.50+/-0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34+/-0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA-/QA redox potential of -0.08 V, the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 mus); -0.42 V for Phe-/Phe; -0.58 V for P680*/P680+.     

一种酿酒酵母附加型分泌表达载体pYES2/CT/α-factor的构建及鉴定

以pPIC9为模板,通过PCR扩增获得酿酒酵母的α-交配因子（α-factor）,并克隆至酿酒酵母胞内表达载体pYES2/CT中,构建了一种新型酿酒酵母附加型分泌表达载体pYES2/CT/α-factor（pYCα）。再将甘露聚糖酶基因（mannase,man）通过酶切、连接克隆至pYCα的α-factor的下游,构建了pYCα-man重组载体检验pYCα的分泌性能和稳定性。结果显示α-factor可引导甘露聚糖酶基因在胞外分泌表达,在曲利本兰培养基上形成明显的水解圈,进一步分析重组菌胞外和胞内酶活,结果显示对照INVSc1/pYCα的两种酶活都未检测到,而INVSc1/pYCα-man具有明显的胞外酶活,未检测到胞内酶活,说明构建的pYCα具有良好的分泌性能;稳定性实验表明重组质粒连续培养150 h仍具有良好的稳定性。     

Nanosecond fluorescence from chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.     

Immunoglobulin allotypes (GM and KM) indicate multiple founding populations of Native Americans: evidence of at least four migrations to the New World.

On the basis of GM and KM typing and language, approximately 28,000 Amerindians were divided into 4 groups of populations: non-Nadene South American (8 groups), non-Nadene North American (7 groups), Nadene (4 groups), and Eskaleuts (6 groups). These groups were compared to four groups of Asian populations. The distribution of GM haplotypes differed significantly among and within these groups as measured by chi-square analysis. Furthermore, as reflected in a maximum linkage cluster analysis, Amerindian populations in general cluster along geographic divisions, with Eskaleuts and Nadenes clustering with the Asian populations and non-Nadene North American and non-Nadene South American populations forming two additional clusters. Based on GM haplotype data and other genetic polymorphisms, the divisions appear to reflect populations that entered the New World at different times. It appears that the South American non-Nadene populations are the oldest, characterized by the haplotypes GM*A G and GM*X G, whereas later North American non-Nadene populations are characterized by high frequencies of GM*A G and low frequencies of GM*X G and GM*A T. In contrast, Eskaleuts appear to have only GM*A G and GM*A T. The Nadene speakers have GM*X G and GM*A T in higher and approximately equal frequencies. Maximum linkage cluster analysis places the Alaskan Athapaskans closest to northwestern Siberian populations and the Eskaleuts closest to the Chukchi, their closest Asian neighbor. These analyses, when combined with other data, suggest that, in the peopling of the New World, at least four separate migrant groups crossed Beringia at various times. It appears likely that the South American non-Nadene entered the New World before 17,000 years B.P. and that the North American non-Nadene entered in the immediate postglacial period, with the Eskaleut and Nadene arriving at a later date.     

Effect of base stacking on the relative thermodynamic stability of oligonucleotide complexes: a spectroscopic study

Three-strand oligonucleotide complexes are employed to assess the effect of base stacking and base pair mismatch on the relative thermodynamic stabilities of oligonucleotide duplexes. The melting behavior of three-strand oligonucleotide complexes incorporating nicks and gaps as well as internal single base mismatches is monitored using temperature-dependent optical absorption spectroscopy. A sequential three-state equilibrium model is used to analyze the measured melting profiles and evaluate thermodynamic parameters associated with dissociation of the complexes. The free-energy of stabilization of a nick complex compared to a gap complex due to base stacking is determined to be -1.9 kcal/mol. The influence of a mispaired base in these systems is shown to destabilize a nick complex by 3.1 kcal/mol and a gap complex by 2.8 kcal/mol, respectively.     

Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.     

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.