Targeted DNA oxidation and trajectory of radical DNA using DFT based QM/MM dynamics.
ABSTRACT: Molecular insight into electronic rearrangements and structural trajectories arising from oxidative damages to DNA backbone is of crucial importance in understanding the effect of ionizing radiation, developing DNA biosensors and designing effective DNA cleaving molecules. Employing a Density Functional Theory based multi-scale Quantum-Mechanical-Molecular-Mechanical (QM/MM) simulation and a suitable partitioning of the Hamiltonian on solvated nucleotide, and single-, and double-stranded DNA, we mimic hydrogen transfer reactions from the backbone by OH radicals and report structural trajectories arising from on-the-fly electronic charge- and spin-density redistribution in these three different structural topologies of DNA. Trajectories reveal that H4' abstraction can disrupt the deoxyribose moiety through the formation of C4'=O4' ketone and a ?-bond with base at C1'-N9 in a nucleotide versus only partial ketone formation in single- and double-stranded DNA, where the orientation of the base is topologically restrained. However, H5' abstraction can lead DNA cleavage at 5' end through the formation of C5'=O5' ketone and breakage of P-O5' bond. Results demonstrate that structural damages from oxidative reactions are restrained by base stacking and base-pair hydrogen bonding. The methodology can be suitably used to study targeted DNA and RNA damages from radicals and radiomimetic drugs to design DNA cleaving molecules for chemotherapy.
Project description:The recently discovered twister ribozyme is thought to utilize general acid-base catalysis in its self-cleavage mechanism, but the roles of nucleobases and metal ions in the mechanism are unclear. Herein, molecular dynamics simulations of the env22 twister ribozyme are performed to elucidate the structural and equilibrium dynamical properties, as well as to examine the role of Mg(2+) ions and possible candidates for the general base and acid in the self-cleavage mechanism. The active site region and the ends of the pseudoknots were found to be less mobile than other regions of the ribozyme, most likely providing structural stability and possibly facilitating catalysis. A purported catalytic Mg(2+) ion and the closest neighboring Mg(2+) ion remained chelated and relatively immobile throughout the microsecond trajectories, although removal of these Mg(2+) ions did not lead to any significant changes in the structure or equilibrium motions of the ribozyme on the microsecond time scale. In addition, a third metal ion, a Na(+) ion remained close to A1(O5'), the leaving group atom, during the majority of the microsecond trajectories, suggesting that it might stabilize the negative charge on A1(O5') during self-cleavage. The locations of these cations and their interactions with key nucleotides in the active site suggest that they may be catalytically relevant. The P1 stem is partially melted at its top and bottom in the crystal structure and further unwinds in the trajectories. The simulations also revealed an interconnected network comprised of hydrogen-bonding and ?-stacking interactions that create a relatively rigid network around the self-cleavage site. The nucleotides involved in this network are among the highly conserved nucleotides in twister ribozymes, suggesting that this interaction network may be important to structure and function.
Project description:Ionizing radiation produces clustered lesions in DNA. Since the orientation of bistranded lesions affects their recognition by DNA repair enzymes, clustered damages are more difficult to process and thus more toxic than single oxidative lesions. In order to understand the structural determinants that lead to differential recognition, we used NMR spectroscopy and restrained molecular dynamics to solve the structure of two DNA duplexes, each containing two stable abasic site analogues positioned on opposite strands of the duplex and staggered in the 3' (-1 duplex, (AP) 2-1 duplex) or 5' (+1 duplex, (AP) 2+1 duplex) direction. Cross-peak connectivities observed in the nonexchangeable NOESY spectra indicate compression of the helix at the lesion site of the duplexes, resulting in the formation of two abasic bulges. The exchangeable proton spectra show the AP site partner nucleotides forming interstrand hydrogen bonds that are characteristic of a Watson-Crick G.C base pairs, confirming the extra helical nature of the AP residues. Restrained molecular dynamics simulations generate a set of converging structures in full agreement with the spectroscopic data. In the (AP) 2-1 duplex, the extra helical abasic site residues reside in the minor groove of the helix, while they appear in the major groove in the (AP) 2+1 duplex. These structural differences are consistent with the differential recognition of bistranded abasic site lesions by human AP endonuclease.
Project description:The solution structure of the 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2R,3R-butanediol cross-link arising from N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) was determined in the oligodeoxynucleotide 5'-d(CGGACXYGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5'-side of the cross-link there was a break in Watson-Crick base pairing at base pair X(6).T(17), whereas at the 3'-side of the cross-link at base pair Y(7).T(16), base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from (1)H NOESY data and 151 torsion angle restraints obtained from (1)H and (31)P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X(6).T(17), accompanied by a shift in the phosphodiester backbone torsion angle beta P5'-O5'-C5'-C4' at nucleotide X(6). The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the beta-carbon of the four-carbon cross-link and T(17) O(4), which perhaps stabilized the base pair opening at X(6).T(17) and protected the T(17) imino proton from solvent exchange. The opening of base pair X(6).T(17) altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].
Project description:Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.
Project description:The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report the crystal structure of a pistol ribozyme termed env25, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2'-O of G for attack on the adjacent to-be-cleaved P-O5' bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2'-OH positions) are aligned to act as a general base and a general acid, respectively, to accelerate cleavage chemistry, with their roles confirmed by cleavage assays on variants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defined how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on variants revealing key residues that participate in acid-base-catalyzed cleavage chemistry.
Project description:Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.
Project description:S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a beta-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.
Project description:DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.
Project description:The incorporation of 6-thioguanine (S6G) into DNA is an essential step in the cytotoxic activity of thiopurines. However, the structural effects of this substitution on duplex DNA have not been fully characterized. Here, we present the solution structures of DNA duplexes containing S6G opposite thymine (S6G.T) and opposite cytosine (S6G.C), solved by high-resolution NMR spectroscopy and restrained molecular dynamics. The data indicate that both duplexes adopt right-handed helical conformations with all Watson-Crick hydrogen bonding in place. The S6G.T structures exhibit a wobble-type base pairing at the lesion site, with thymine shifted toward the major groove and S6G displaced toward the minor groove. Aside from the lesion site, the helices, including the flanking base pairs, are not highly perturbed by the presence of the lesion. Surprisingly, thermal dependence experiments suggest greater stability in the S6G-T mismatch than the S6G-C base pair.
Project description:Thymidylate synthase (TS) catalyzes the substitution of a carbon-bound proton in a uracil base by a methyl group to yield thymine in the de novo biosynthesis of this DNA base. The enzymatic mechanism involves making and breaking several covalent bonds. Traditionally, a conserved tyrosine (Y94 in Escherichia coli, Y146 in Lactobacillus casei, and Y135 in humans) was assumed to serve as the general base catalyzing the proton abstraction. That assumption was examined here by comparing the nature of the proton abstraction using wild-type (wt) E. coli TS (ecTS) and its Y94F mutant (with a turnover rate reduced by 2 orders of magnitude). A subsequent hydride transfer was also studied using the wt and Y94F. The physical nature of both H-transfer steps was examined by determining intrinsic kinetic isotope effects (KIEs). Surprisingly, the findings did not suggest a direct role for Y94 in the proton abstraction step. The effect of this mutation on the subsequent hydride transfer was examined by a comparison of the temperature dependency of the intrinsic KIE on both the wt and the mutant. The intrinsic KIEs for Y94F at physiological temperatures were slightly smaller than those for wt but, otherwise, were as temperature-independent, suggesting a perfectly preorganized reaction coordinate for both enzymes. At reduced temperatures, however, the KIE for the mutant increased with a decrease in temperature, indicating a poorly preorganized reaction coordinate. Other kinetic and structural properties were also compared, and the findings suggested that Y94 is part of a H-bond network that plays a critical role at a step between the proton and the hydride transfers, presumably the dissociation of H4folate from the covalently bound intermediate. The possibility that no single residue serves as the general base in question but, rather, that the whole network of H-bonds at the active site catalyzes proton abstraction is discussed.