Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes and they can damage biological molecules including nucleic acids. It was shown that X-or γ-ray irradiation of aqueous solutions of DNA, during which · OH is one of the major ROS, can lead to the formation of intrastrand crosslink lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32 P-postlabeling studies suggested that the intrastrand crosslink lesions may arise from Fenton reaction, which also induces the formation of · OH; the structures of the proposed intrastrand crosslink lesions, however, have not been determined. Here we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H 2 O 2 /ascorbate could lead to the formation of an intrastrand crosslink lesion, i.e., G^T, where the C8 of guanine is covalently bonded to the neighboring 3′ thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G^T. In addition, the yield of the intrastrand crosslink was approximately three orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2′-deoxyguanosine, 5-(hydroxymethyl)-2′-deoxyuridine and 5-formyl-2′-deoxyuridine. The induction of intrastrand crosslink lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.

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Molecular dynamics simulations of fluoroalkyl-derivatized imidazolium:bis(trifluoromethylsulfonyl)imide (TFSI) room temperature ionic liquids (FADI-RTILs) with cations of the structure 1-F(CF(2))(n)(CH(2))(2)-3-methyl imidazolium have been performed and compared with simulations of alkyl-derivatized 1-H(CH(2))(n+2)-3-methyl imidazolium analogs (ADI-RTILs). Simulations yield RTIL densities, viscosities and ionic conductivities for the FADI-RTILs and ADI-RTILs in reasonably good agreement with experimental data. Partial fluorination results in a larger increase in density than would be anticipated based upon the density difference between perfluoralkane and alkane melts. Similarly, the slowing down in dynamics upon partial fluorination is greater than would be expected based upon the increase in cation volume. Examination of cation-cation, anion-anion and cation-anion centers-of-mass radial distribution functions reveal remarkably little influence of partial fluorination on the spherically averaged intermolecular structure of the RTILs. Similarly, simulations reveal little change in tail conformations and the extent of tail-tail aggregation upon partial fluorination. The interaction of the TFSI anion with the positively charged imidazolium ring hydrogen and nitrogen atoms is also little influenced by partial fluorination. However, the partially fluorinated alkyl tail exhibits increased interaction with the TFSI anion due to the electron withdrawing character of the fluorinated groups. We believe this strong tail-anion electrostatic interaction largely accounts for the higher than expected density and slower than expected dynamics in the FADI-RTILs.