Structure and Stability of Duplex DNA Containing (5′S)-5′,8-Cyclo-2′-deoxyadenosine: An Oxidatively Generated Lesion Repaired by NER (original) (raw)
Related papers
2012
Cellular respiration and ionizing radiation generate 5′,8-cyclo-2′-deoxyribonucleosides, a special type of DNA damage that involves two modifications in the same nucleotide. These lesions evade the action of base excision glycosylases, and their removal is a function of the nucleotide excision repair pathway. Diastereomeric 5′,8-cyclo-2′-deoxyadenosine blocks mammalian DNA replication, diminishes the levels of DNA transcription, and induces transcriptional mutagenesis. Using solution state NMR spectroscopy and restrained molecular dynamics simulations, we have determined the structure of an undecameric DNA duplex having a centrally located (5′S)-5′,8-cyclo-2′-deoxyadenosine residue paired to T. The damaged duplex structure is a right-handed helix having Watson−Crick base-pair alignments throughout, and 2-deoxyribose puckers within the B-form conformation. Only small structural perturbations are observed at the lesion-containing and 5′-flanking base pair. The 2-deoxyribose of the damaged nucleotide adopts the O4′-exo conformation, and the S-cdA•T base pair is propeller twisted. The 5′-lesion-flanking base is tilted forming a significantly buckled base pair with its partner guanine. Analysis of UV-melting curves indicates mild thermal and thermodynamic destabilization on the damaged duplex. The S-cdA•T duplex structure shows many similarities to and some intriguing differences from the recently reported structure of an S-cdG•dC duplex 31 that suggest different lesion site dynamics.
Journal of The Royal Society Interface, 2008
We have used molecular dynamics simulations to study the structure and dynamics of a range of DNA duplexes containing the 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapydG) lesion that can result from oxidative damage at guanine. Compared to the corresponding undamaged DNA duplexes, FapydG-containing duplexes show little gross structural changes-the damaged base remains stacked in to the DNA double helix and retains hydrogen bonds to its cytosine partner. However, the experimentally observed reduction in DNA stability that accompanies lesion formation can be explained by a careful energetic analysis of the simulation data. Irrespective of the nature of the base pairs on either side of the lesion site, conversion of a guanine to a FapydG base results in increased dynamical flexibility in the base (but not in the DNA as a whole) that significantly weakens its hydrogen-bonding interactions. Surprisingly, the stacking interactions with its neighbours are not greatly altered. The formamido group adopts a non-planar conformation that can interact significantly and in a sequence-dependent manner with its 3 0 -neighbour. We conclude that the recognition of FapydG lesions by the repair protein formamidopyrimidine-DNA glycosylase probably does not involve the protein capturing an already-extrahelical FapydG base, but rather it relies on detecting alterations to the DNA structure and flexibility created by the lesion site.
Biopolymers, 2015
The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (T g) lesion on a family of deoxyoligonucleotide duplexes. T g damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The T g lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates T g-induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-T g isomer centered within the tridecanucleotide sequence 5 0-GCGTACXCATGCG-3 0 (X 5 T g or T) which is hybridized with the corresponding complementary sequence 5 0-CGCATGNGTACGC-3 0 (N 5 A, G, T, C) to generate families of T g-damaged (T g ÁN) and lesion-free (TÁN) duplexes. We demonstrate that the magnitude and nature of the T g destabilizing impact is dependent on counterbase identity (i.e., A $ G < T < C). The observation that a T g lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of T g ÁN duplexes parallel their respective lesion-free TÁN mismatch counterparts (i.e., G < T < C). Elucidation of T g-induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The T g-induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery.
Biopolymers, 2018
Bistrand lesions embedded within a single helical turn of tridecameric deoxyoligonucleotide duplexes represent a model system for exploring the impact of clustered lesions that occur in vivo and pose a significant challenge to cellular repair machineries. Such investigations are essential for understanding the forces that dictate lesion-induced mutagenesis, carcinogenesis, and cytotoxicity within a context that mimics local helical perturbations caused by an ionizing radiation event. This study characterizes the structural and energy profiles of DNA duplexes harboring synthetic abasic sites (tetrahydrofuran, F) as models of clustered bistrand abasic (AP) lesions. The standard tridecameric dGCGTACCCATGCGÁdCGCATGGGTACGC duplex is employed to investigate the energetic impact of single and bistrand AP sites by strategically replacing one or two bases within the central CCC/GGG triplet. Our combined analysis of temperature-dependent UV and circular dichroism (CD) profiles reveals that the proximity and relative orientation of AP sites within bistranddamaged duplexes imparts a significant thermodynamic impact. Specifically, 3 0-staggered lesions (CCF/GFG) exert a greater destabilizing effect when compared with their 5 0-counterpart (FCC/ GFG). Moreover, a duplex harboring the central bistrand AP lesion (CFC/GFG) is moderately destabilized yet exhibits distinct properties relative to both the 3 0 and 5 0-orientations. Collectively, our energetic data are consistent with structural studies on bistrand AP-duplexes of similar sequence in which a 3 0-staggered lesion exerts the greatest perturbation, a finding that provides significant insight regarding the impact of orientation on lesion repair processing efficiency.
Chemical Research in Toxicology, 2000
The T‚G mismatch and the exocyclic adduct 3,N 4 -ethenocytosine ( C) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or C base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T‚G and C-containing 15mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5′-TTXTT, 5′-AAXAA, 5′-CCXCC, or 5′-GGXGG sequences, where X is T, C, or two closely related structural derivatives of C: 3,N 4 -ethanocytosine (EC) and 8-(hydroxymethyl)-C (8-HM-C). Each of the four lesions, incorporated opposite G, decreased both the thermal (T m ) and thermodynamic stability (∆G°3 7 ) of the 15-mer control duplexes. On the basis of the T m and ∆G°3 7 values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-C > EC > C > T‚G. The ∆T m values range from -15.8 to -9.5°C when C t ) 8 µM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2°C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4°C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3.0°C
Biochemistry, 2012
Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2′-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2′-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2′-deoxycytidine·2′-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2′-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.
Solution Structure of Duplex DNA Containing a β‑Carba-Fapy-dG LesionStates *S Supporting Information
The addition of hydroxyl radicals to the C8 position of guanine can lead to the formation of a 2,6-diamino-4-hydroxy-5formamido-2′-deoxypyrimidine (Fapy-dG) lesion, whose endogenous levels in cellular DNA rival those of 8-oxo-7,8-dihydroxy-2′deoxyguanosine. Despite its prevalence, the structure of duplex DNA containing Fapy-dG is unknown. We have prepared an undecameric duplex containing a centrally located β-cFapy-dG residue paired to dC and determined its solution structure by high-resolution NMR spectroscopy and restrained molecular dynamic simulations. The damaged duplex adopts a right-handed helical structure with all residues in an anti conformation, forming Watson−Crick base pair alignments, and 2-deoxyribose conformations in the C2′endo/C1′-exo range. The formamido group of Fapy rotates out of the pyrimidine plane and is present in the Z and E configurations that equilibrate with an approximate 2:1 population ratio. The two isomeric duplexes show similar lesion-induced deviations from a canonical B-from DNA conformation that are minor and limited to the central three-base-pair segment of the duplex, affecting the stacking interactions with the 5-lesion-neighboring residue. We discuss the implications of our observations for translesion synthesis during DNA replication and the recognition of Fapy-dG by DNA glycosylases.
Solution Structure of Duplex DNA Containing a β-Carba-Fapy-dG Lesion
Chemical Research in Toxicology, 2012
The addition of hydroxyl radicals to the C8 position of guanine can lead to the formation of 2,6diamino-4-hydroxy-5-formamido-2′-deoxypyrimidine (Fapy-dG) lesion, whose endogenous levels in cellular DNA rival those of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine. Despite its prevalence, the structure of duplex DNA containing Fapy-dG is unknown. We have prepared an undecameric duplex containing a centrally located β-cFapy-dG residue paired to dC and determined its solution structure by high resolution NMR spectroscopy and restrained molecular dynamic simulations. The damaged duplex adopts a right-handed helical structure all residues in an anti conformation, forming Watson-Crick base pair alignments, and 2-deoxyribose conformations in the C2′-endo/ C1′-exo range. The formamido group of Fapy rotates out of the pyrimidine plane and is present on the Z and E configurations that equilibrate with an approximate 2:1 population ratio. The two isomeric duplexes show similar lesion induced deviations from a canonical B-from DNA conformation that are minor and limited to the central three-base-pair segment of the duplex, affecting the stacking interactions with the 5-lesion-neighboring residue. We discuss the implications of our observations for translesion synthesis during DNA replication and the recognition of Fapy-dG by DNA glycosylases.
2008
The relative biological importance of cis-syn cyclobutane dimer and pyrimidine(6-4)pyrimidone photoadduct ([6-41 photoadduct) appears to be dependent on the biological species, dipyrimidine sites and the local conformational variation induced at the damaged sites. The single-stranded deoxynucleotide 10-mers containing the site-specific (6-4) adduct or cis-sytz cyclobutane dimer of thymidylyl (3'-+5')-thymidine were generated by direct photolysis of d(CGCATTACGC) with UVC (220-260 nm) irradiation or UVB (260-320 nm) photosensitization. Three-dimensional structures of the duplex cissyn and (6-4) decamers of d(CGCATTACGC).d(GCGTAATGCG) were determined by NMR spectroscopy and the relaxation matrix refinement method. The NMR data and structural calculations establish that Watson-Crick base pairing is still intact at the cis-syn dimer site while the hydrogen bonding is absent at the 3'-side of the (6-4) lesion where the T + C transition mutation is predominantly targeted. Overall conformation of the duplex cis-syn decamer was B-DNA and produced a 9" bending in the DNA helix, but a distinctive base orientation of the (6-4) lesion provided a structural basis leading to 44" helical bending. The observed local structure and conformational rigidity at the (6-4) adduct of the thymidylyl(3'-5')-thymidine (T-T [6-41) lesion site suggest the potential absence of hydrogen bonding at the 3' sides o f the (6-4) lesion with a substituted nucleotide during replication under S O S conditions. Contrasting structural distortions induced by the T-T (6-4) adduct with respect to the T-T cis-syn cyclobutane pyrimidine photodimer may explain the large differences in mutation spectrum and repair activities between them
Nucleic Acids Research, 2002
Two series of duplex DNA oligomers were prepared having an anthraquinone derivative (AQ) covalently linked at a 5′-terminus. Irradiation of the AQ at 350 nm leads to injection of an electron hole (radical cation) into the DNA. The radical cation migrates through the DNA causing reaction primarily at G n sequences. In one series, GA tandem mispairs are inserted between GG steps to assess the effect of the mispair on the transport of the radical cation, reaction (damage) caused by the radical cation at the mispair, and repair of the resulting damage by formamidopyrimidine DNA glycosylase (Fpg). In the second series, a bulged guanine in a G 3 C 2 sequence is interposed between the GG steps. These experiments reveal that neither G/A tandem mispairs nor bulged guanines are significant barriers to long-range charge migration in DNA. The radical cation does not cause reaction at guanines in the G/A tandem mispair. Reaction does occur at the bulged guanine, but it is repaired by Fpg.