Electron paramagnetic resonance evidence for a C3' sugar radical in crystalline d(CTCTCGAGAG) X-irradiated at 4 K

Debije, M. G. and Bernhard, W. A. Electron Paramagnetic Resonance Evidence for a C3' Sugar Radical in Crystalline d(CTCTCGAGAG) X-Irradiated at 4 K. Radiat. Res. 155, 687-692 (2001). A neutral sugar radical formed by the net loss of hydrogen from C3' has been identified in crystalline DNA X-irradiated at 4 K. Crystals of duplex d(CTCTCGAGAG), known to be of B conformation, were studied using electron paramagnetic resonance (EPR) spectroscopy. The C3' radical was identified by using information from dose saturation, power saturation, thermal annealing, and spectrum simulation. The yield of the C3' radical, G(C3'), is 0.03 +/- 0.01 micromol/J, and its concentration does not appear to saturate up to at least 100 kGy. In the region in which total radical concentration increases linearly with dose, the C3' radical makes up about 4.5% of the total radical population trapped in the oligodeoxynucleotide crystal at 4 K. Based on free base release measured in other oligodeoxynucleotides, we suggest that in d(CTCTCGAGAG) the C3' radical is responsible for about one-third of the strand breakage events.     

Unaltered free base release from d(CGCGCG)2 produced by the direct effect of ionizing radiation at 4 K and room temperature

Unaltered free base release in d(CGCGCG)2 exposed to X rays at 4 K or room temperature was measured by HPLC. Samples were prepared either as films hydrated to a level of Gamma = 2.5 mol water/mol nucleotide or as polycrystalline with Gamma approximately 7.5 mol water/mol nucleotide. X irradiation of films at 4 K, followed by annealing to room temperature, resulted in yields for cytosine and guanine of G(Cyt) = 0.036 +/- 0.001 micromol/J and G(Gua) = 0.090 +/- 0.002 micromol/J. Irradiation of films at room temperature gave similar yields. The yields for polycrystalline d(CGCGCG)2 X-irradiated at room temperature were G(Cyt) = 0.035 +/- 0.005 micromol/J and G(Gua) = 0.077 +/- 0.023 micromol/J. The total free base release yield, G(fbr), was 0.124 +/- 0.008 micromol/J for films and 0.112 +/- 0.028 micromol/J for polycrystalline samples. G(fbr) is believed to be a good estimate of total strand break yield. The yields of total free radicals trapped [G(Sigmafr)] by the d(CGCGCG)2 films at 4 K were measured by EPR. The measured value, G(Sigmafr) = 0.450 +/- 0.005 micromol/J, was used to calculate the yield of trappable sugar radicals, giving G(sugar)(fr) = 0.04-0.07 micromol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) G(sugar)(fr) cannot account for more than half of the free base release, and (3) G(fbr), G(Cyt) and G(Gua) were independent of the sample temperature during irradiation. Finding (1) suggests that base and or sequence influences sugar damage, and finding (2) is consistent with our working hypothesis that an important pathway to strand break formation entails two one-electron oxidations at the same sugar site.     

Direct radiation damage to crystalline DNA: what is the source of unaltered base release?

The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 micromol J(-1). The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient.     

Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

Yields of damage to C4' deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation

Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the (32)P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991) (32)P-Postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry, 30, 1091-1097] which when applied to the indirect effect, detected phosphoglycolate (pg) and thymine glycol (Tg). Here we applied this assay to the direct effect, measuring product yields in pUC18 films with hydration levels (Γ) of 2.5, 16 or 23 waters per nucleotide and X-irradiated at either 4 K or room temperature (RT). The yields of pg [G(pg)] for Γ ≈ 2.5 were 2.8 ± 0.2 nmol/J (RT) and 0.2 ± 0.3 nmol/J (4 K), which is evidence that the C4' radical contributes little to the total deoxyribose damage via the direct effect. The yield of detectable base damage [G(B*)] at Γ ≈ 2.5 was found to be 30.2 ± 1.0 nmol/J (RT) and 12.9 ± 0.7 nmol/J (4 K). While the base damage called B*, could be due to either oxidation or reduction, we argue that two reduction products, 5,6-dihydrouracil and 5,6-dihydrothymine, are the most likely candidates.     

On the efficiency of hole and electron transfer from the hydration layer to DNA: An EPR study of crystalline DNA X-irradiated at 4 K

The aim of this project was to gain an improved understanding of how the efficiency of hole and electron transfer from the solvation layer to DNA decreases as a function of distance from DNA. The packing of DNA in crystals of known structure makes it possible to calculate the degree of DNA hydration with a precision that is significantly greater than that achievable for amorphous samples. Previous work on oligodeoxynucleotide crystals has demonstrated that the efficiency of free radical trapping by DNA exposed to ionizing radiation at 4 K is relatively insensitive to base sequence, conformation, counterion, or base stacking continuity. Having eliminated these confounding variables, it is now possible to ascertain the degree of radical transfer that occurs from ionized water as a function of DNA hydration (Gamma, in mol water/mol nucleotide). EPR is used to measure the hydroxyl radical concentration in crystals irradiated at 4 K. From a lack of hydroxyl radicals trapped in the inner hydration mantle, we determine that hole transfer to DNA is complete for water molecules located within 8 A. This corresponds to Gamma = 9-11 and indicates that hole transfer is 100% (as efficient as direct ionization of DNA) for water molecules adjacent to DNA. Beyond approximately 8 A (Gamma > 10), hydroxyl radicals are observed; thus deprotonation of the water radical cation is seen to compete with hole transfer to DNA as soon as one water intervenes between the ionized water and DNA. The boundary for 0% hole transfer is projected to occur somewhere between 15 and 20 waters per nucleotide. Electron transfer, on the other hand, is 100% efficient across the entire range studied, 4.2 相似文献   

Electron and hole transfer from DNA base radicals to oxidized products of guanine in DNA

An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.     

Mechanisms of direct radiation damage in DNA, based on a study of the yields of base damage, deoxyribose damage, and trapped radicals in d(GCACGCGTGC)(2)

Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.     

Radiation-induced free radicals in solid 5-halouracil derivatives: single crystals of 5-iododeoxyuridine

X-irradiation of single crystals of 5-iododeoxyuridine (IUdR) in the temperature range 8-300 K produces mainly four different radicals which have been studied by electron spin resonance (e.s.r.) and electron nuclear double resonance (ENDOR)-spectroscopy. At low temperatures, a pi-anion is formed which shows predominantly an interaction of the unpaired electron with a proton at carbon C6 of the base (-11.8 G, -23.9 G, -4.6 G). Above 10-20 K, the anion protonates at C6 to yield a RC-I(CH2)-R' radical comprising alpha-iodo and beta-methylene proton hyperfine interactions. The primary oxidation product is an O5'-situated alkoxy radical RCH2O which shows inequivalent beta-proton couplings of about 100 G and 35 G together with a highly anisotropic g-tensor. Upon warming to 265 K, a C2'-located radical on the deoxyribose is formed which is stable at room temperature. A detailed account of its spectral features as obtained by ENDOR exhibits three different alpha-type couplings, two small beta-protons and a dipolar interaction. Other radicals, not reproducibly observed, involve a C5'-hydroxyalkyl radical and a species related to the base cation at low temperatures.     

Structures of Escherichia coli DNA mismatch repair enzyme MutS in complex with different mismatches: a common recognition mode for diverse substrates

We have refined a series of isomorphous crystal structures of the Escherichia coli DNA mismatch repair enzyme MutS in complex with G:T, A:A, C:A and G:G mismatches and also with a single unpaired thymidine. In all these structures, the DNA is kinked by ~60° upon protein binding. Two residues widely conserved in the MutS family are involved in mismatch recognition. The phenylalanine, Phe 36, is seen stacking on one of the mismatched bases. The same base is also seen forming a hydrogen bond to the glutamate Glu 38. This hydrogen bond involves the N7 if the base stacking on Phe 36 is a purine and the N3 if it is a pyrimidine (thymine). Thus, MutS uses a common binding mode to recognize a wide range of mismatches.     

The formation of DNA sugar radicals from photoexcitation of guanine cation radicals

In this investigation of radical formation and reaction in gamma- irradiated DNA and model compounds, we report the conversion of the guanine cation radical (one-electron oxidized guanine, G(.+)) to the C1' sugar radical and another sugar radical at the C3' or C4' position (designated C3'(.)/C4'(.)) by visible and UV photolysis. Electron spin resonance (ESR) spectroscopic investigations were performed on salmon testes DNA as well as 5'-dGMP, 3'-dGMP, 2'-deoxyguanosine and other nucleosides/nucleotides as model systems. DNA samples (25- 150 mg/ml D(2)O) were prepared with Tl(3+) or Fe(CN)(3-)(6) as electron scavengers. Upon gamma irradiation of such samples at 77 K, the electron-gain path in the DNA is strongly suppressed and predominantly G(.+) is found; after UV or visible photolysis, the fraction of the C1' sugar radical increases with a concomitant reduction in the fraction of G(.+). In model systems, 3'- dGMP(+.) and 5'-dGMP(+.) were produced by attack of Cl(.-)(2) on the parent nucleotide in 7 M LiCl glass. Subsequent visible photolysis of the 3'-dGMP(+.) (77 K) results predominantly in formation of C1'(.) whereas photolysis of 5'-dGMP(+.) results predominantly in formation of C3'(.)/C4'(.). We propose that sugar radical formation is a result of delocalization of the hole in the electronically excited base cation radical into the sugar ring, followed by deprotonation at specific sites on the sugar.     

Recognition of base J in duplex DNA by J-binding protein.

beta-d-Glucosylhydroxymethyluracil, also called base J, is an unusual modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously found a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia. We have now characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contain the glycosylated base in various DNA sequences. We find that JBP recognizes base J only when presented in double-stranded DNA but not in single-stranded DNA or in an RNA:DNA duplex. It also fails to interact with free glucose or free base J. JBP is unable to recognize nonmodified DNA or intermediates of J synthesis, suggesting that JBP is not directly involved in J biosynthesis. JBP binds J-DNA with high affinity (K(d) = 40-140 nm) but requires at least 5 bp flanking the glycosylated base for optimal binding. The nature of the flanking sequence affects binding because J in a telomeric sequence binds JBP with higher affinity than J in another sequence known to contain J in trypanosome DNA. We conclude that JBP is a structure-specific DNA-binding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.     

The effects of ionizing radiation on DNA: the role of thiols as radioprotectors

Electron loss from N-(2-mercaptopropionyl) glycine (PSH) gave an EPR detectable radical anion, PS-.SP(-). When the PSH derivative was frozen in aqueous DNA solutions to 77 K and exposed to ionizing radiation, normal damage to the DNA was detected by EPR spectroscopy. However, on annealing above 77 K, central EPR features for the DNA base radical cations and anions gave central features assigned to PS-.SP(-) sigma*-radical anions, together with outer features for 5-6-dihydro-5-thymyl radicals, TH.. It is proposed that on freezing, the PSH molecules are constrained into a glassy region around the DNA, and that, on annealing, electron donation gives PS. radicals, with loss of quanine radical-cations, G(.+). The PS. radicals were not detectable, but on reaction with another PSH molecule, gave good EPR spectra for PS-.SP(-) radical-anions. These results indicate that PSH had little effect on the yield of the other base radicals C(.-)/T(.-). Also, growth of TH. radicals, formed from protonated thymine radical-anions, T(.-), were detected. We conclude that the primary effect of PSH is to capture the G(.+) centers, and thus could either prevent or repair radiation damage to DNA.     

Stability of DNA duplexes with Watson-Crick base pairs: a predicted model

The conformational stability (difference between the free energies of the folded and unfolded states, DeltaG degrees ) of a DNA duplex is considered as a function of component energy terms, hydrophobic, base stacking, hydrogen bonding, van der Waals, and electrostatic, and a trinucleotide-level helix stiffness parameter measured in terms of its Young's modulus. Hydrophobic and base stacking energy components were determined with the use of the crystal structure data of 30 DNA duplexes judicially selected within a resolution of 1.5 A, and hydrogen bonding, van der Waals and electrostatic terms were determined through an extensive review of experimental and theoretical studies. The stiffness indices for the trinucleotides were the ones realized by M. M. Gromiha [(2000) J. Biol. Phys. 26, 43-50] using the crystal structure data of 70 DNA duplexes. The unfolded state was treated in the classical way to determine its stability. Thermodynamically determined DeltaG degrees values for 111 DNA duplexes, with the number of base pairs ranging from 4 to 16, were selected in two sets, and the regression equation formed with one set was used to predict the stabilities of the other set, taking the energy components and the stiffness parameter to be independent variables. The computed energy terms indicate that the base stacking and hydrogen bonding forces are the dominant and the hydrophobic and electrostatic forces the weak partners in imparting stability to the duplexes. This model predicts DeltaG degrees values for DNA duplexes examined with a level of accuracy similar to that used for predictions made by the widely used nearest-neighbor models. The uniqueness of this model is that it combines the crystal and thermodynamic data for interpretation of conformational stability.     

Electron spin resonance study of DNA irradiated with an argon-ion beam: evidence for formation of sugar phosphate backbone radicals

In this study, the effects of high-LET radiation on DNA were investigated and compared with the effects of gamma radiation. Hydrated DNA samples at 77 K were irradiated with argon-ion beams ((36)Ar or (40)Ar beam at energies between 60 and 100 MeV/nucleon). The individual free radicals formed were identified and their yields were investigated by electron spin resonance spectroscopy. Argon-ion irradiation resulted in lower yields of base ion radicals and higher yields of neutral radicals than gamma irradiation. A hitherto unknown species was assigned to the radical formed by C-O bond rupture at the deoxyribose C3', resulting in a sugar carbon-centered radical. A previously characterized phosphorus-centered radical was also found. The formation of each of these species was accompanied by an immediate strand break. G values, k values, and analyses for the individual yields of neutral radicals and ion radical composition for argon-ion-irradiated hydrated DNA are reported and compared to those found previously for gamma-irradiated DNA. The lower G values and k values for ion radicals and the higher fraction of neutral radicals found for argon-ion-irradiated DNA are attributed to differences in track structure inherent in the two radiations.     

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.     

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.     

Local conformational variations observed in B-DNA crystals do not improve base stacking: computational analysis of base stacking in a d(CATGGGCCCATG)(2) B<-->A intermediate crystal structure

The crystal structure of d(CATGGGCCCATG)₂ shows unique stacking patterns of a stable B↔A-DNA intermediate. We evaluated intrinsic base stacking energies in this crystal structure using an ab initio quantum mechanical method. We found that all crystal base pair steps have stacking energies close to their values in the standard and crystal B-DNA geometries. Thus, naturally occurring stacking geometries were essentially isoenergetic while individual base pair steps differed substantially in the balance of intra-strand and inter-strand stacking terms. Also, relative dispersion, electrostatic and polarization contributions to the stability of different base pair steps were very sensitive to base composition and sequence context. A large stacking flexibility is most apparent for the CpA step, while the GpG step is characterized by weak intra-strand stacking. Hydration effects were estimated using the Langevin dipoles solvation model. These calculations showed that an aqueous environment efficiently compensates for electrostatic stacking contributions. Finally, we have carried out explicit solvent molecular dynamics simulation of the d(CATGGGCCCATG)₂ duplex in water. Here the DNA conformation did not retain the initial crystal geometry, but moved from the B↔A intermediate towards the B-DNA structure. The base stacking energy improved in the course of this simulation. Our findings indicate that intrinsic base stacking interactions are not sufficient to stabilize the local conformational variations in crystals.     

Multiplicity of DNA single-strand breaks produced in pUC18 exposed to the direct effects of ionizing radiation

The transition of plasmid DNA from a supercoiled to an open circle conformation, as detected by gel electrophoresis, affords an extraordinarily sensitive method for detecting single-strand breaks (SSBs), one measure of deoxyribose damage. To determine the yield of SSBs, G(ssb), by this method, it is commonly assumed that Poisson statistics apply such that, on average, one SSB occurs per supercoiled plasmid lost. For the direct effect, at a large enough plasmid size, this assumption may be invalid. In this report, the assumption that one SSB occurs per pUC18 plasmid (2686 bp) is tested by measuring free base release (fbr), which is also a measure of deoxyribose damage in films prepared under controlled relative humidity so as to produce known levels of DNA hydration. The level of DNA hydration, Gamma, is expressed in mol water/mol nucleotide. The yield of free base release, G(fbr), was measured by HPLC after exposure of the films to 70 kV X rays and subsequent dissolution in water. It is well known that damage in deoxyribose leads to SSBs and free base release. Based on known mechanisms, there exists a close correspondence between free base release and SSBs, i.e., G(fbr) congruent with G(ssb). Following this assumption, the SSB multiplicity, m(ssb), was determined, where m(ssb) was defined as the mean number of SSBs per supercoiled plasmid lost. The yield of lost supercoil was determined previously (S. Purkayastha et al., J. Phys. Chem. B 110, 26286-26291, 2006). We found that m(ssb) = 1.4 +/- 0.2 at Gamma = 2.5 and m(ssb) = 2.8 +/- 0.5 to 3.1 +/- 0.5 at Gamma = 22.5, indicating that the assumption of one SSB per lost supercoil is not likely to hold for a 2686-bp plasmid exposed to the direct effect. In addition, an increase in G(fbr), upon stepping from Gamma = 2.5 to Gamma = 22.5, was paralleled by an increase in the yield of trapped deoxyribose radicals, G(dRib)(fr), also measured previously. As a consequence, the shortfall between SSBs and trapped radicals, G(diff) = G(ssb) - G(dRib)(fr), remained relatively constant at 90-110 nmol/J. The lack of change between the two extremes of hydration is in keeping with the suggestion that non-radical species, such as doubly oxidized deoxyribose, are responsible for the shortfall.     

Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.