On the molecular mechanism of drug intercalation into DNA: a simulation study of the intercalation pathway, free energy, and DNA structural changes

Intercalation into DNA (insertion between a pair of base pairs) is a critical step in the function of many anticancer drugs. Despite its importance, a detailed mechanistic understanding of this process at the molecular level is lacking. We have constructed, using extensive atomistic computer simulations and umbrella sampling techniques, a free energy landscape for the intercalation of the anticancer drug daunomycin into a twelve base pair B-DNA. A similar free energy landscape has been constructed for a probable intermediate DNA minor groove-bound state. These allow a molecular level understanding of aspects of the thermodynamics, DNA structural changes, and kinetic pathways of the intercalation process. Key DNA structural changes involve opening the future intercalation site base pairs toward the minor groove (positive roll), followed by an increase in the rise, accompanied by hydrogen bonding changes of the minor groove waters. The calculated intercalation free energy change is -12.3 kcal/mol, in reasonable agreement with the experimental estimate -9.4 kcal/mol. The results point to a mechanism in which the drug first binds to the minor groove and then intercalates into the DNA in an activated process, which is found to be in general agreement with experimental kinetic results.     

Interconversion of the cis-5R,6S- and trans-5R,6R-thymine glycol lesions in duplex DNA

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5R Tg lesion was incorporated site-specifically into 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'; Tg = 5R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)A(19)C(20)G(21)C(22)A(23)C(24))-3', forming the Tg(6) x A(19) base pair, corresponding to the oxidative damage of thymine in DNA, or 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)G(19)C(20)G(21)C(22)A(23)C(24))-3', forming the mismatched Tg(6) x G(19) base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA. At 30 degrees C, the equilibrium ratio of cis-5R,6S:trans-5R,6R epimers was 7:3 for the duplex containing the Tg(6) x A (19) base pair. In contrast, for the duplex containing the Tg(6) x G(19) base pair, the cis-5R,6S:trans-5R,6R equilibrium favored the cis-5R,6S epimer; the level of the trans-5R,6R epimer remained below the level of detection by NMR. The data suggested that Tg disrupted hydrogen bonding interactions, either when placed opposite to A(19) or G(19). Thermodynamic measurements indicated a 13 degrees C reduction of T(m) regardless of whether Tg was placed opposite dG or dA in the complementary strand. Although both pairings increased the free energy of melting by 3 kcal/mol, the melting of the Tg x G pair was more enthalpically favored than was the melting of the Tg x A pair. The observation that the position of the equilibrium between the cis-5R,6S and trans-5R,6R thymine glycol epimers in duplex DNA was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of Tg [Ocampo-Hafalla, M. T.; Altamirano, A.; Basu, A. K.; Chan, M. K.; Ocampo, J. E.; Cummings, A., Jr.; Boorstein, R. J.; Cunningham, R. P.; Teebor, G. W. DNA Repair (Amst) 2006, 5, 444-454].     

Entropy of water in the hydration layer of major and minor grooves of DNA

Transport properties (translational and rotational) of water in the two grooves of the B-DNA duplex are known to be different from those in the bulk. Here, we use a recently developed theoretical scheme to compute the entropies of water molecules in both of the grooves of DNA and compare them with that in the bulk. The scheme requires as input both translational and rotational velocity autocorrelation function (C(V)(t) and C(omega)(t), respectively) data. These velocity autocorrelation functions were computed from an atomistic MD simulation of a B-DNA duplex (36 base pairs long) in explicit water (TIP3P). The average values of the entropy of water at 300 K in both of the grooves of DNA (the TS value in the major groove is 6.71 kcal/mol and that in the minor groove is 6.41 kcal/mol) are found to be significantly lower than that in bulk water (the TS value is 7.27 kcal/mol). Thus, the entropic contribution to the free energy change (TDeltaS) of transferring a minor groove water molecule to the bulk is 0.86 kcal/mol and of transferring a major groove water to the bulk is 0.56 kcal/mol at 300 K, which is to be compared with 1.44 kcal/mol for melting of ice at 273 K. We also calculate the energy of interaction of each water molecule with the rest of the atoms in the system and hence calculate the chemical potential (Helmholtz free energy per water molecule, A = E - TS) in the different domains. The identical free energy value of water molecules in the different domains proves the robustness of the scheme. We propose that the configurational entropy of water in the grooves can be used as a measure of the mobility (or microviscosity) of water molecules in a given domain.     

Docking, triggering, and biological activity of dynemicin A in DNA: a computational study

The triggering and biological activity of the naturally occurring enediyne dynemicin A (1) was investigated, both inside and outside the minor groove of the duplex 10-mer B-DNA sequence d(CTACTACTGG).d(CCAGTAGTAG), using density functional theory (B3LYP with the 3-21G and 6-31G(d) basis set), BD(T)/cc-pVDZ (Brueckner doubles with a perturbative treatment of triple excitations), and the ONIOM approach. Enediyne 1 is triggered by NADPH in a strongly exothermic reaction (-88 kcal/mol), which involves a number of intermediate steps. Untriggered 1 has a high barrier for the Bergman cyclization (52 kcal/mol) that is lowered after triggering to 16.7 kcal/mol due to an epoxide opening and the accompanying strain relief. The Bergman reaction of triggered 1 is slightly exothermic by 2.8 kcal/mol. The singlet biradical formed in this reaction is kinetically stable (activation enthalpies of 19.5 and 21.8 kcal/mol for retro-Bergman reactions) and is as reactive as para-benzyne. The activity-relevant docking mode is an edge-on insertion into the minor groove, whereas the intercalation between base pairs, although leading to larger binding energies, excludes a triggering of 1 and the development of its biological activity. Therefore, an insertion-intercalation model is developed, which can explain all known experimental observations made for 1. On the basis of the insertion-intercalation model it is explained why large intercalation energies suppress the biological activity of dynemicin and why double-strand scission can be achieved only in a two-step mechanism that involves two enediyne molecules, explaining thus the high ratio of single-strand to double-strand scission observed for 1.     

Reactions of 1,3-cyclohexadiene with singlet oxygen. A theoretical study.

A thorough study of the reaction of singlet oxygen with 1,3-cyclohexadiene has been made at the B3LYP/6-31G(d) and CASPT2(12e,10o) levels. The initial addition reaction follows a stepwise diradical pathway to form cyclohexadiene endoperoxide with an activation barrier of 6.5 kcal/mol (standard level = CASPT2(12e,10o)/6-31G(d); geometries and zero-point corrections at B3LYP/6-31G(d)), which is consistent with an experimental value of 5.5 kcal/mol. However, as the enthalpy of the transition structure for the second step is lower than the diradical intermediate, the reaction might also be viewed as a nonsynchronous concerted reaction. In fact, the concertedness of the reaction is temperature dependent since entropy differences create a free energy barrier for the second step of 1.8 kcal/mol at 298 K. There are two ene reactions; one is a concerted mechanism (DeltaH(double dagger) = 8.8 kcal/mol) to 1-hydroperoxy-2,5-cyclohexadiene (5), while the other, which forms 1-hydroperoxy-2,4-cyclohexadiene (18), passes through the same diradical intermediate (9) as found on the pathway to endoperoxide. The major pathway from the endoperoxide is O-O bond cleavage (22.0 kcal/mol barrier) to form a 1,4-diradical (25), which is 13.9 kcal/mol less stable than the endoperoxide. From the diradical, two low-energy pathways exist, one to epoxyketone (29) and the other to the diepoxide (27), where both products are known to be formed experimentally with a product ratio sensitive to the nature of substitutents. A significantly higher activation barrier leads to C-C bond cleavage and direct formation of maleic aldehyde plus ethylene.     

The mechanisms of the reactions of W and W+ with COx (x=1, 2): a computational study

The mechanisms of the reactions of W and W+ with COx (x=1, 2) were studied at the CCSD(T)/[SDD+6-311G(d)]//B3LYP/[SDD+6-31G(d)] level of theory. It was shown that the gas-phase reaction of W with CO2 proceeds with a negligible barrier via an insertion pathway, W(7S)+CO2(1A1)-->W(eta2-OCO)(6A')-->OW(eta1-CO)(1A)-->WO (3Sigma+)+CO(1Sigma). This oxidation process is calculated to be exothermic by 32.4 kcal/mol. Possible intermediates of this reaction are the W(eta2-OCO) and OWCO complexes, among which the latter is 37.4 kcal/mol more stable and lies 39.7 and 7.3 kcal/mol lower than the reactants, W(7S)+CO2(1A1), and the products, WO (3Sigma+)+CO(1Sigma), respectively. The barrier separating W(eta2-OCO) from OWCO is 8.0 kcal/mol (relative to the W(eta2-OCO) complex), which may be characterized as a W+delta-(CO2)-delta charge-transfer complex. Ionization of W does not change the character of the reaction of W with CO2: the reaction of W+ with CO2, like its neutral analog, proceeds via an insertion pathway and leads to oxidation of the W-center. The overall reaction W+(6D) + CO2(1A1)-->W(eta1-OCO)+(6A)-->OW(eta1-CO)+(4A)-->WO+(4Sigma+)+CO(1Sigma) is calculated to be exothermic by 25.4 kcal/mol. The cationic reaction proceeds with a somewhat large (9.9 kcal/mol) barrier and produces two intermediates, W(eta1-OCO)+(6A) and OW(eta1-CO)+(4A). Intermediate W(eta1-OCO)+(6A) is 20.0 kcal/mol less stable than OW(eta1-CO)+(4A), and separated from the latter by a 35.2 kcal/mol barrier. Complex W(eta1-OCO)+(6A) is characterized as an ion-molecular complex type of W+-(CO2). Gas-phase reactions of M=W/W+ with CO lead to the formation of a W-carbonyl complex M(eta1-CO) for both M=W and W+. The C-O insertion product, OMC, lies by 5.2 and 69.3 kcal/mol higher than the corresponding M(eta1-CO) isomer, for M=W and W+, respectively, and is separated from the latter by a large energy barrier.     

Disproportionation of Gold(II) complexes. A density functional study of ligand and solvent effects

A computational study of gold(II) disproportionation is presented for the atomic ion as well as complexes with chloride and neutral ligands. The Au2+ atomic ion is stable to disproportionation, but the barrier is more than halved to 119 kcal/mol in an aqueous environment vs 283 kcal/mol in the gas phase. For dissociative disproportionation of chloride complexes, the loss of chlorine, either as an atom (Delta G(aq) = +20 kcal/mol) or as an anion (Delta G(aq) = +15 kcal/mol) represents the largest calculated barrier. The calculated transition state for associative disproportionation is only 9 kcal/mol above separated Au(II)Cl3(-) anions. For the disproportionation of Au(II)L3 complexes with neutral ligands, disproportionation is highly endergonic in the gas phase. Calculations imply that for synthesis of a monometallic Au(II) complex, a nonpolar solvent is preferred. With the exception of [Au(CO)3]2+, disproportionation of Au(II)L3 complexes to Au(I)L and Au(III)L3 is exergonic in solution phase for the ligands investigated. The driving force is provided by the very favorable solvation free energy of the trivalent gold complex. The solvation free energy contribution to the reaction (Delta G(solv)) is very large for small and polar ligands such as ammonia and water. Furthermore, calculations imply that choosing ligands that would yield neutral species upon disproportionation may provide an effective route to thwart this decomposition pathway for Au(II) complexes. Likewise, bulkier ligands that yield larger, more weakly solvated complex ions would appear to be desirable.     

Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines.

A heptamer composed of C5-(1-propynyl) pyrimidines (Y(p)'s) is a potent and specific antisense agent against the mRNA of SV40 large T antigen (Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.; Froehler, B. C. Nat. Biotechnol. 1996, 14, 840-844). To characterize the role of the propynyl groups in molecular recognition, thermodynamic increments associated with substitutions in DNA:RNA duplexes, such as 5'-dCCUCCUU-3':3'-rGAGGAGGAAAU-5', have been measured by UV melting experiments. For nucleotides tested, an unpaired dangling end stabilizes unmodified and propynylated duplexes similarly, except that addition of a 5' unpaired rA is 1.4 kcal/mol more stabilizing on the propynylated, PODN:RNA, duplex than on the DNA:RNA duplex. Free energy increments for addition of single propynyl groups range from 0 to -4.0 kcal/mol, depending on the final number and locations of substitutions. A preliminary model for predicting the stabilities of Y(p)-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5'-dC(p)C(p)U(p)C(p)C(p)U(p)U(p)-3':3'-rGAGIAGGAAAU-5', destabilizes the duplex by 3.9 kcal/mol, compared to 1.7 kcal/mol for the same change within the unpropynylated duplex. This 2.2 kcal/mol difference is eliminated by removing a single propynyl group three base pairs away. CD spectra suggest that single propynyl deletions within the PODN:RNA duplex have position-dependent effects on helix geometry. The results suggest long-range cooperativity between propynyl groups and provide insights for rationally programming oligonucleotides with enhanced binding and specificity. This can be exploited in developing technologies that are dependent upon nucleic acid-based molecular recognition.     

Azole-bridged diplatinum anticancer compounds. Modulating DNA flexibility to escape repair mechanism and avoid cross resistance

Dinuclear azole-bridged Pt compounds bind to DNA helices, forming intrastrand crosslinks between adjacent guanines in a similar way to cisplatin. Their cytotoxic profile is, however, different from that of first and second generation Pt drugs in that they lack cross resistance in cisplatin-resistant cell lines. In contrast to cisplatin, which induces a large kink in DNA duplex, structural NMR studies and molecular dynamics simulations have shown that azole-bridged diplatinum compounds induce only small structural changes in double-stranded DNA. These structural differences have been invoked to explain the different cytotoxic profile of these compounds. Here, we show that in addition to the small structural changes in DNA, dinuclear Pt compounds also affect DNA minor groove flexibility in a different way than cisplatin. Free-energy calculations on azole-bridged diplatinum DNA adducts reveal that opening of the minor groove requires a higher free-energy cost (DeltaG ~ 7-15 kcal/mol) than in the corresponding cisplatin-DNA adduct (DeltaG ~ 0 kcal/mol). This could prevent minor groove binding proteins from binding to diplatinum-DNA adducts thus leading to a different cellular response than cisplatin and possibly decreasing the activity of excision repair enzymes. Although the development of drug resistance is a highly complex mechanism, our findings provide an additional rationale for the improved cytotoxic activity of these compounds in cell lines resistant to cisplatin.     

Ab initio and RRKM study of the HCN/HNC elimination channels from vinyl cyanide

Ab initio CCSD and CCSD(T) calculations with the 6-311+G(2d,2p) and the 6-311++G(3df,3pd) basis sets were carried out to characterize the vinyl cyanide (C(3)H(3)N) dissociation channels leading to hydrogen cyanide (HCN) and its isomer hydrogen isocyanide (HNC). Our computations predict three elimination channels giving rise to HCN and another four channels leading to HNC formation. The relative HCN/HNC branching ratios as a function of internal energy of vinyl cyanide were computed using RRKM theory and the kinetic Monte Carlo method. At low internal energies (120 kcal/mol), the total HCN/HNC ratio is about 14, but at 148 kcal/mol (193 nm) this ratio becomes 1.9, in contrast with the value 124 obtained in a previous ab initio/RRKM study at 193 nm (Derecskei-Kovacs, A.; North, S. W. J. Chem. Phys.1999, 110, 2862). Moreover, our theoretical results predict a ratio of rovibrationally excited acetylene over total acetylene of 3.3, in perfect agreement with very recent experimental measurements (Wilhelm, M. J.; Nikow, M.; Letendre, L.; Dai, H.-L. J. Chem. Phys.2009, 130, 044307).     

Quantum chemical study of low temperature oxidation mechanism of dibenzofuran

A density functional theory (DFT) study of the reaction of dibenzofuranyl radical with oxygen molecule has been made. The geometries, energies, and vibrational frequencies of the reactant, transition states, intermediates, and products have been calculated at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31G(d) level of theory. The initial reaction of dibenzofuran (DBF) with molecular oxygen results in the formation of the 1-dibenzofuranylperoxy radical. The stability of this adduct toward decomposition at low to intermediate temperatures results in it undergoing several possible rearrangements. The lowest energy pathway with a barrier of 24.2 kcal/mol involves a rearrangement to the 1,1-dioxadibenzofuran radical. The next lowest energy pathway involves fission of the O-O linkage whose reaction energy was found to be 37.6 kcal/mol. Transition state theory (TST) calculations indicate that the lowest energy pathway should predominate at temperatures up to about 1200 K. Two other unimolecular reaction pathways with barriers of 45.5 and 91.1 kcal/mol have also been discovered. The latter pathway leads to the formation of a para-quinone (dibenzofuran quinone) which has been detected experimentally in the low-temperature oxidation of DBF [Marquaire, P. M.; Worner, R.; Rambaud, P.; Baronnet, F. Organohalogen Compd. 1999, 40, 519]. Our quantum calculations, however, do not support this latter pathway to quinone formation. Instead, the quinone is most probably formed as a consequence of recombination of the 1-dibenzofuranyloxy radical (produced by peroxy fission) with an O atom in the para position. Each of the unimolecular reaction pathways have been subjected to detailed quantum chemical investigation and transition states and intermediates leading to the final products (principally CO, CO2, and C2H2 with traces of benzofuran and benzene) have been identified. For certain stable intermediates, their possible reactions with molecular oxygen have been further investigated quantum chemically. The present work therefore presents a detailed quantum chemical investigation of the reaction pathways in the low-temperature oxidation mechanism of DBF. Since the dibenzofuran moiety is present in the polychlorinated DBFs, our conclusions should be generally applicable to this family of compounds.     

Computational study of sulfur atom-transfer reactions from thiiranes to ER3 (E = As, P; R = CH3, Ph)

Computational estimates have been made for the P=S and As=S bond strengths in triphenylphosphine sulfide and triphenylarsine sulfide, on the basis of G3 calculations for the methyl analogues and isodesmic-exchange reactions. Also, with the performance of the G3 method level for related compounds taken into consideration, the best estimates are 82 and 68 kcal/mol, respectively. While the value for triphenylarsine sulfide is within 2 kcal/mol of the single experimental estimate, that for triphenylphosphine sulfide is lower by 6 kcal/mol. (Capps, K. B.; Wixmerten, B.; Bauer, A.; Hoff, C. D. Inorg. Chem. 1998, 37, 2861-2864.) Despite virtually identical electronegativities of P and As, it is found that there is greater charge separation in the P=S bond. It is found that S atom transfer from thiiranes to arsines is exothermic.     

Reaction of ethylene with hydroxyl radicals: a theoretical study

Ab initio calculations of portions of the C2H5O potential energy surface critical to the title reaction are presented. These calculations are based on QCISD geometries and frequencies and RQCISD(T) energies extrapolated to the complete-basis-set limit. Rate coefficients for the reaction of C2H4 with OH are calculated using this surface and the two transition-state model of Greenwald and co-workers [J. Phys. Chem. A 2005, 109, 6031] for the association of OH with C2H4. The present calculations reproduce most of the experimental data, including the temperature and pressure dependence of the rate coefficients, with only a small (0.4 kcal/mol) adjustment to the energy barrier for direct hydrogen abstraction. We confirm the importance of this channel above 800 K and find that a significant fraction of the total rate coefficient (approximately 10%) is due to the formation of vinyl alcohol above this temperature. Calculations of the vinyl alcohol channel are consistent with the recent observation of this molecule in low-pressure flames [Taatjes, C. A.; Hansen, N.; McIlroy, A.; Miller, J. A.; Senosiain, J. P.; Klippenstein, S. J.; Qi, F.; Sheng, L.; Zhang, Y.; Cool, T. A.; Wang, J.; Westmoreland, P. R.; Law, M. E.; Kasper, T.; Kohse-H?inghaus, K. Science 2005, 308, 1887] and suggest that this reaction should be included in hydrocarbon oxidation mechanisms.     

Mechanisms of the reactions of W and W+ with NOx (x=1, 2): a computational study

The mechanisms of the reactions of W and W+ with NOx (x=1, 2) were studied at the CCSD(T)/[SDD+6-311G(d)]//B3LYP/[SDD+6-31G(d)] level of theory. It was shown that the insertion pathway of the reaction W(7S)+NO2(2A1) is a multistate process, which involves several lower lying electronic states of numerous intermediates and transition states, and leads to oxidation, WO(3Sigma)+NO(2Pi), and/or nitration, WN(4Sigma)+O2(3Sigmag-), of the W-center. Oxidation products WO(3Sigma)+NO(2Pi) lie 87.6 kcal/mol below the reactants, while the nitration channel is only 31.0 kcal/mol exothermic. Furthermore, it was shown that nitration of W with NO2 is kinetically less favorable than its oxidation. The addition-dissociation pathway of the reaction W(7S)+NO2(2A1) proceeds via the octet (ground) state potential energy surface of the reaction, requires 3.3 kcal/mol barrier, and leads exclusively to oxidation products. Calculations show that oxidation of the W+ cation by NO2 is a barrierless process in the gas phase, proceeds exclusively via the insertion pathway, and is exothermic by 82.9 kcal/mol. The nitration of W+ by NO2 is only 14.1 kcal/mol exothermic and could be accessible only under high-temperature conditions. Reactions of M=W/W+ with NO are also barrierless processes in the gas phase and lead to the N-O insertion product NMO, which are 105.4 and 77.4 kcal/mol lower than the reactants for W and W+, respectively.     

Computational evaluation of the evidence for tri-trans-[12]annulene

[reaction: see text] Automerization in tri-trans-[12]annulene (1) was investigated by DFT, MP2, and coupled-cluster methods. Using the highest level of theory employed here, CCSD(T)/cc-pVDZ//BHandHLYP/6-311+G(d,p), we located two low-energy pathways for degenerate conformational change from the lowest-energy conformer of 1 (1a): one with E(a) = 4.5 kcal/mol that interconverts the three inner trans hydrogens with the three outer trans hydrogens and one with E(a) = 2.7 kcal/mol that interconverts the three inner hydrogens with each other. These results are consistent with the experimental results of Oth and co-workers on [12]annulene 1a (Oth, J. F. M.; R?ttele, H.; Schr?der, G. Tetrahedron Lett. 1970, 61). The conformational exchange of the inner trans hydrogens with the outer ones is predicted to occur via a one-step process involving a C(2)-symmetric transition state and not via the D(3)-symmetric transition state (1b) that was postulated earlier. Conformer 1b was found to be a shallow minimum 6.7 kcal/mol above 1a with a barrier of 0.4 kcal/mol for conversion to 1a. Finally, GIAO-B3LYP/6-311+G(d,p) and BHandHLYP/6-311+G(d,p) computed (1)H NMR chemical shifts of 1a and three other low-lying isomers support Oth's original assignment of observed (1)H NMR peaks to 1a at both low and high temperature.     

Alteration in the choice of DNA repair pathway with increasing sequence selective DNA alkylation in the minor groove

BACKGROUND: Many conventional DNA alkylating anticancer drugs form adducts in the major groove of DNA. These are known to be chiefly repaired by both nucleotide (NER) and base (BER) excision repair in eukaryotic cells. Much less is known about the repair pathways acting on sequence specific minor groove purine adducts, which result from a promising new class of anti-tumour agents. RESULTS: Benzoic acid mustards (BAMs) tethering 1-3 pyrrole units (compounds 1, 2 and 3) show increasing DNA sequence selectivity for alkylation from BAM and 1, alkylating primarily at guanine-N7 in the major groove, to 3 which is selective for alkylation in the minor groove at purine-N3 in the sequence 5'-TTTTGPu (Pu=guanine or adenine). This increasing sequence selectivity is reflected in increased toxicity in human cells. In the yeast Saccharomyces cerevisiae, the repair of untargeted DNA adducts produced by BAM, 1 and 2 depends upon both the NER and BER pathways. In contrast, the repair of the sequence specific minor groove adducts of 3 does not involve known BER or NER activities. In addition, neither recombination nor mismatch repair are involved. Two disruptants from the RAD6 mutagenesis defective epistasis group (rad6 and rad18), however, showed increased sensitivity to 3. In particular, the rad18 mutant was over three orders of magnitude more sensitive to 3 compared to its isogenic parent, and 3 was highly mutagenic in the absence of RAD18. Elimination of the sequence specific DNA adducts formed by 3 was observed in the wild type strain, but these lesions persisted in the rad18 mutant. CONCLUSIONS: We have demonstrated that the repair of DNA adducts produced by the highly sequence specific minor groove alkylating agent 3 involves an error free adduct elimination pathway dependent on the Rad18 protein. This represents the first systematic analysis of the cellular pathways which modulate sensitivity to this new class of DNA sequence specific drugs, and indicates that the enhanced cytotoxicity of certain sequence specific minor groove adducts in DNA is the result of evasion of the common excision repair pathways.     

Aqueous solvation free energies of ions and ion-water clusters based on an accurate value for the absolute aqueous solvation free energy of the proton

Thermochemical cycles that involve pKa, gas-phase acidities, aqueous solvation free energies of neutral species, and gas-phase clustering free energies have been used with the cluster pair approximation to determine the absolute aqueous solvation free energy of the proton. The best value obtained in this work is in good agreement with the value reported by Tissandier et al. (Tissandier, M. D.; Cowen, K. A.; Feng, W. Y.; Gundlach, E.; Cohen, M. J.; Earhart, A. D.; Coe, J. V. J. Phys. Chem. A 1998, 102, 7787), who applied the cluster pair approximation to a less diverse and smaller data set of ions. We agree with previous workers who advocated the value of -265.9 kcal/mol for the absolute aqueous solvation free energy of the proton. Considering the uncertainties associated with the experimental gas-phase free energies of ions that are required to use the cluster pair approximation as well as analyses of various subsets of data, we estimate an uncertainty for the absolute aqueous solvation free energy of the proton of no less than 2 kcal/mol. Using a value of -265.9 kcal/mol for the absolute aqueous solvation free energy of the proton, we expand and update our previous compilation of absolute aqueous solvation free energies; this new data set contains conventional and absolute aqueous solvation free energies for 121 unclustered ions (not including the proton) and 147 conventional and absolute aqueous solvation free energies for 51 clustered ions containing from 1 to 6 water molecules. When tested against the same set of ions that was recently used to develop the SM6 continuum solvation model, SM6 retains its previously determined high accuracy; indeed, in most cases the mean unsigned error improves when it is tested against the more accurate reference data.     

Free energy simulations of uncatalyzed DNA replication fidelity: structure and stability of T.G and dTTP.G terminal DNA mismatches flanked by a single dangling nucleotide

A reference system for DNA replication fidelity was studied by free energy perturbation (FEP) and linear interaction energy (LIE) methods. The studied system included a hydrated duplex DNA with the 5'-CG dangling end of the templating strand, and dCTP4-.Mg2+ or dTTP4-.Mg2+ inserted opposite the dangling G to form a correct (i.e., Watson-Crick) or incorrect (i.e., wobble) base pair, respectively. The average distance between the 3'-terminal oxygen of the primer strand and the alpha-phosphorus of dNTP was found to be 0.2 A shorter for the correct base pair than for the incorrect base pair. Binding of the incorrect dNTP was found to be disfavored by 0.4 kcal/mol relative to the correct dNTP. We estimated that improved binding and more near-attack configurations sampled by the correct base pair should translate in aqueous solution and in the absence of DNA polymerase into a six times faster rate for the incorporation of the correct dNTP into DNA. The accuracy of the calculated binding free energy difference was verified by examining the relative free energy for melting duplex DNA containing GC and GT terminal base pairs flanked by a 5' dangling C. The calculated LIE and FEP free energies of 1.7 and 1.1 kcal/mol, respectively, compared favorably with the experimental estimate of 1.4 kcal/mol obtained using the nearest neighbor parameters. To decompose the calculated free energies into additive electrostatic and van der Waals contributions and to provide a set of rigorous theoretical data for the parametrization of the LIE method, we suggested a variant of the FEP approach, for which we coined a binding-relevant free energy (BRFE) acronym. BRFE approach is characterized by its unique perturbation pathway and by its exclusion of the intramolecular energy of a rigid part of the ligand from the total potential energy.