Abasic Site Lesions Research Articles

C. elegans ribosomal protein S3 protects against H2O2-induced DNA damage and suppresses spontaneous mutations in yeast

Carcinogenicity and cytotoxicity are severe consequences of DNA damage. Base Excision Repair (BER) is a conserved DNA repair pathway that replaces many damaged bases caused by oxidation. Aberrations in BER are associated with carcinogenesis, neurodegeneration, and aging. The nematode C. elegans is an attractive model system for studying BER. However, in this organism, the complete pathway is not fully delineated. To further explore the BER process in C. elegans, we used affinity tag chromatography and mass spectrometry to identify the interactome of uracil DNA glycosylase-1 (CeUNG-1), an enzyme that acts during the first step of the BER pathway. Our analysis identified that CeUNG-1 is associated with the 40 S ribosomal protein S3 (CeRPS-3), homologs of which have been shown to process 8-oxoguanine and abasic site lesions in other organisms. We report a strong in silico association between CeUNG-1 and CeRPS-3 and confirmed this interaction using the yeast two-hybrid system. Downregulation of the Cerps-3 gene reduced the viability of wild-type worms upon exposure to the chemical oxidant hydrogen peroxide. Further analysis shows that Cerps-3 knockdown significantly sensitized the AP endonuclease APN-1-deficient strain, apn-1, but to a lesser extent exo-3, to the lethal effects of hydrogen peroxide. A cross-species complementation experiment reveals that the expression of CeRPS-3 rescued the hydrogen peroxide sensitivity, and suppressed the high mutation frequency of the yeast AP endonuclease-deficient strain lacking Apn1 and Apn2. We propose that CeRPS-3 may function as an auxiliary DNA repair enzyme in C. elegans to process oxidative DNA lesions.

Bypass of an Abasic Site via the A-Rule by DNA Polymerase of Pseudomonas aeruginosa Phage PaP1.

The abasic site is one the most common DNA lesions formed in cells; it induces a severe blockage of DNA replication and is highly mutagenic. We continue to use Gp90 exo-, the sole DNA polymerase from Pseudomonas aeruginosa phage PaP1, to study DNA replication upon encountering an abasic site lesion. Gp90 exo- can incorporate dNTPs opposite the abasic site, but extension past this site is extremely slow. Among the four dNTPs, dATP is preferentially incorporated opposite the abasic site, consistent with the A-rule. The incorporation is independent of the identity of the nucleotide 5' of the abasic site. The incorporation of dATP opposite the abasic site occurs by direct incorporation of dNTP opposite the abasic site without a -1 frameshift deletion. Extension from an A:abasic site pair by Gp90 exo- is slightly unfavorable relative to those from other abasic site pairs. Incorporation of dATP opposite the abasic site is preferential and shows a biphasic shape, indicating that this incorporation is much faster than the subsequent dissociation of the polymerase from DNA. The template sequence does not affect the dATP incorporation priority, burst amplitude, burst rate, or dATP dissociation constant. Surface plasmon resonance shows that the presence of an abasic site in the template weakens the binding affinity of Gp90 exo- to DNA in a binary or ternary complex in the presence of any one kind of dNTP. This study reveals that Gp90 exo- preferentially inserts A opposite an abasic site via the A-rule, like other DNA polymerases (e.g., Pol θ, KlenTaq, KF exo-, Pols α, δ/PCNA, and Thermococcus litoralis Pol Vent (exo-)), providing further insight into DNA replication mediated by P. aeruginosa phage PaP1 upon encountering an abasic site lesion.

Impeded repair of abasic site damaged lesions in DNA adsorbed over functionalized multiwalled carbon nanotube and graphene oxide

The processing of abasic site DNA damage lesions in extracellular DNA in the presence of engineered carbon nanomaterials (CNMs) is demonstrated. The efficacy of the apurinic-apyrimidinic endonuclease 1 (APE1) in the cleavage of abasic site lesions in the presence of carboxylated multi-walled carbon nanotubes (MWCNT-COOH) and graphene oxide (GO) are compared. The CNMs were found to perturb the incision activity of APE1. The reason for such perturbation process was anticipated to take place either by the non-specific adsorption of APE1 over the free surface of the CNMs or steric hindrance offered by the CNM-DNA complex. Accordingly, bovine serum albumin (BSA) was selectively utilized to block the free surface of the CNM-DNA hybrid material. Further treatment of the CNM-DNA-BSA complex with APE1 resulted in a marginal increase in APE1 efficiency. This indicates that APE1 in solution is unable to process the abasic sites on DNA adsorbed over the CNMs. However, the cleavage activity of APE1 was restored in the presence of non-ionic surfactant (Tween 20) that inhibits adsorption of the DNA on the surface of the CNMs. The conformational deformation of the DNA, along with steric hindrance induced by the CNMs resulted in the inhibition of abasic site DNA repair by APE1. Moreover, appreciable changes in the secondary structure of APE1 adsorbed over the CNMs were observed that contribute further to the repair refractivity of the abasic sites. From a toxicological viewpoint, these findings can be extended to the study of the effect of engineered nanoparticles in the intracellular DNA repair process.

DNA containing a cluster of 8-oxo-guanine and abasic site lesion : alpha anomer (AP6, 8OG 14)

DNA containing a cluster of 8-oxo-guanine and abasic site lesion : alpha anomer (AP6, 8OG 14)

Structure and Dynamics of DNA Duplexes Containing a Cluster of Mutagenic 8-Oxoguanine and Abasic Site Lesions

Clustered DNA damage sites are caused by ionizing radiation. They are much more difficult to repair than are isolated single lesions, and their biological outcomes in terms of mutagenesis and repair inhibition are strongly dependent on the type, relative position and orientation of the lesions present in the cluster. To determine whether these effects on repair mechanism could be due to local structural properties within DNA, we used 1H NMR spectroscopy and restrained molecular dynamics simulation to elucidate the structures of three DNA duplexes containing bistranded clusters of lesions. Each DNA sequence contained an abasic site in the middle of one strand and differed by the relative position of the 8-oxoguanine, staggered on either the 3′ or the 5′ side of the complementary strand. Their repair by base excision repair protein Fpg was either complete or inhibited. All the studied damaged DNA duplexes adopt an overall B-form conformation and the damaged residues remain intrahelical. No striking deformations of the DNA chain have been observed as a result of close proximity of the lesions. These results rule out the possibility that differential recognition of clustered DNA lesions by the Fpg protein could be due to changes in the DNA's structural features induced by those lesions and provide new insight into the Fpg recognition process.

The abasic site lesions in the human telomeric sequence d[TA(G3T2A)3G3]: A thermodynamic point of view

BackgroundThe abasic sites represent one of the most frequent lesions of DNA and most of the events able to generate such modifications involve guanine bases. G-rich sequences are able to form quadruplex structures that have been proved to be involved in several important biological processes. MethodsIn this paper, we report investigations, based on calorimetric, UV, CD and electrophoretic techniques, on 12 oligodeoxynucleotides analogues of the quadruplex forming human telomere sequence d[TA(G3T2A)3G3], in which each guanine has been replaced, one at a time, by an abasic site mimic. ResultsAlthough all data show that the modified sequences preserve their ability to form quadruplex structures, the thermodynamic parameters clearly indicate that the presence of an abasic site decreases their thermal stability compared to the parent unmodified sequence, particularly if the replacement concerns one of the guanosines involved in the formation of the central G-tetrad. ConclusionsThe collected data indicate that the effects of the presence of abasic site lesions in telomeric quadruplex structures are site-specific. The most dramatic consequences come out when this lesion involves a guanosine in the centre of a G-run. General significanceAbasic sites, by facilitating the G-quadruplex disruption, could favour the formation of the telomerase primer. Furthermore they could have implications in the pharmacological approach targeting telomere.

NMR Solution Structures of Clustered Abasic Site Lesions in DNA: Structural Differences between 3′-Staggered (−3) and 5′-Staggered (+3) Bistranded Lesions

Ionizing radiation produces a distinctive pattern of bistranded clustered lesions in DNA. A relatively low number of clustered lesions may be lethal to cells when compared to a larger number of single lesions. Enzyme cleavage experiments suggest that the orientation of bistranded lesions causes differential recognition and removal of these lesions. Like that of a previous study of bistranded abasic site lesion [Hazel, R. D., Tian, K., and de los Santos, C. (2008) Biochemistry 47, 11909-11919], the aim of this investigation was to determine the structures of two DNA duplexes each containing two synthetic apurinic/apyrimidinic (AP) residues, positioned on opposite strands and separated by two base pairs. In the first duplex, the AP residues are staggered in the 3' orientation [-3 duplex, (AP)(2)-3 duplex], while in the second duplex, the AP residues are staggered in the 5' orientation [+3 duplex, (AP)(2)+3 duplex]. NOESY spectra recorded in 100 and 10% D(2)O buffer solutions allowed the assignment of the nonexchangeable and exchangeable protons, respectively, for each duplex. Cross-peak connectivity in the nonexchangeable proton spectra indicates that the duplex is a regular right-handed helix with the AP residues and orphan bases located inside the duplexes. The exchangeable proton spectra establish the formation of Watson-Crick G·C alignment for the two base pairs between the lesion sites in both duplexes. Distance-restrained molecular dynamics simulation confirmed the intrahelical orientations of the AP residues. The proximity of the AP residues across the minor groove of the -3 duplex and across the major groove in the +3 duplex is similar to their locations in the case of -1 and +1 clusters. This difference in structure may be a key factor in the differential recognition of bistranded AP lesions by human AP endonuclease.

Reversible Chemical Step and Rate-Limiting Enzyme Regeneration in the Reaction Catalyzed by Formamidopyrimidine-DNA Glycosylase

Formamidopyrimidine-DNA N-glycosylase (Fpg) operates in the base excision repair pathway in bacteria by removing oxidized guanine bases from DNA and can also cleave the nascent or preformed abasic DNA by beta,delta-elimination. In this work, we have used the quench-flow technique (i) to show that the kinetics of processing of 7,8-dihydro-8-oxoguanine and abasic site lesions by Fpg from Escherichia coli involves a burst phase and a stationary phase, (ii) to establish the reaction kinetic scheme, and (iii) to calculate the rate constants for the reaction steps. A comparison of the quench-flow results with the data from earlier stopped-flow kinetics with tryptophan and 2-aminopurine fluorescence detection reveals that the cleaved product formation is initially reversible; it is followed by conformational changes in the enzyme and DNA molecules that represent the postchemical irreversible rate-limiting steps. We have applied mass spectrometry with electrospray ionization to follow the appearance and disappearance of transient covalent intermediates between Fpg and the substrate DNA. The overall rate-limiting step of the enzymatic reaction seems to be the release of Fpg from its adduct with the 4-oxo-2-pentenal remnant of the deoxyribose moiety formed as a result of DNA strand cleavage by beta,delta-elmination.

Electrostatic potential maps of damaged DNA studied by image analysis tools. 8-Oxoguanine and abasic site lesions

Changes of electrostatic potential around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. The electrostatic potential around the DNA fragments containing either the intact guanine-cytosine pair or 8-oxoguanine-cytosine or the guanine-abasic site was projected on a cylindrical surface around the double helix. The 2D maps of EP of intact and damaged DNA fragments were compared using image analysis methods. Occurrence of abasic site and 8-oxoguanine lesions were found to be reflected in the EP maps. In the case of the 8-oxoguanine lesion, the two phosphate groups and countercations of the damaged strand are moved away from the lesion in opposite directions, whereas they are moved in the same direction in the case of the abasic site lesion. The characteristic features of 8-oxoguanine lesion might be identified in the major groove, whereas the features of abasic site lesion the minor groove.

NMR Solution Structures of Bistranded Abasic Site Lesions in DNA

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.

Effects of Abasic Site and 8oxoG Lesions on DNA Molecule

Effects of Abasic Site and 8oxoG Lesions on DNA Molecule

Effects of Abasic Site and 8oxoG Lesions on DNA Molecule

Effects of Abasic Site and 8oxoG Lesions on DNA Molecule

The Reactivity of the 5-Hydroxy-5,6-dihydrothymidin-6-yl Radical in Oligodeoxyribonucleotides

Hydroxyl radical can be induced from Fenton reaction or gamma radiolysis of water. It can add preferentially to the C5 position of thymidine to give the 5-hydroxy-5,6-dihydrothymidin-6-yl radical. In this report, we examined the independent generation and reactivity of this radical in oligodeoxyribonucleotides (ODNs). Our results showed that the major products originated from this radical in single- and double-stranded ODNs were thymidine glycol, 5-hydroxy-5,6-dihydrothymidine, thymidine, and abasic site lesion. A cross-linking lesion, where the C6 of 5-hydroxy-5,6-dihydrothymidine and the C8 of its neighboring guanine are covalently bonded, could be induced from the independently generated radical in dinucleoside monophosphate and trinucleoside diphosphates. The formation of this type of cross-link product in duplex ODNs from either the independently generated radical or gamma irradiation was near or below the detection limit of the LC-MS/MS method that we used.

Independent generation of the 5-hydroxy-5,6-dihydrothymidin-6-yl radical and its reactivity in dinucleoside monophosphates.

Hydroxyl radical is a major reactive oxygen species produced by gamma-radiolysis of water or Fenton reaction. It attacks pyrimidine bases and gives the 5-hydroxy-5,6-dihydropyrimidin-6-yl radical as the major product. Here we report the synthesis of all four stereoisomers of 5-hydroxy-6-phenylthio-5,6-dihydrothymidine (T*), which, upon 254 nm UV irradiation, give rise to the 5-hydroxy-5,6-dihydrothymidin-6-yl radical (I). We also incorporated the photolabile radical precursors into dinucleoside monophosphates d(GT*) and d(TT*) and characterized major products resulting from the 254-nm irradiation of these dinucleoside monophosphates. Our results showed that, under anaerobic conditions, the most abundant product emanating from the 254-nm irradiation of d(GT*) and d(TT*) is an abasic site lesion. Products with the thymine portion being modified to thymine glycol and 5-hydroxy-5,6-dihydrothymine were also observed. In addition, we demonstrated that radical I can attack the C8 carbon atom of its 5' neighboring guanine and give rise to a novel cross-link lesion. Moreover, LC-MS/MS results showed that gamma-radiation of d(GT) under anaerobic condition yielded the same type of cross-link lesions.

Half-life and DNA strand scission products of 2-deoxyribonolactone oxidative DNA damage lesions.

Reactive oxygen species lead to oxidative damage of the nucleobase and sugar components of nucleotides in double-stranded DNA. The 2-deoxyribonolactone (or oxidized abasic site) lesion results from oxidation of the C-1' position of DNA nucleotides and has been implicated in DNA strand scission, mutagenesis, and covalent cross-linking to DNA binding proteins. We previously described a strategy for the synthesis of DNA-containing deoxyribonolactone lesions. We now report an improved method for the site specific photochemical generation of deoxyribonolactone sites within DNA oligonucleotides and utilize these synthetic oligonucleotides to characterize the products and rates of DNA strand scission at the lactone lesion under simulated physiological conditions. A C-1' nitroveratryl cyanohydrin phosphoramidite analogue was synthesized and used for the preparation of DNA containing a photochemically "caged" lactone precursor. Irradiation at 350 nm quantitatively converted the caged analogue into the deoxyribonolactone lesion. The methodology was validated by RP-HPLC and MALDI-TOF mass spectrometry. Incubation of deoxyribonolactone-containing DNA under simulated physiological conditions gave rise to DNA fragmentation by two consecutive elimination reactions. The DNA-containing products resulting from DNA cleavage at the deoxyribonolactone site were isolated by PAGE and unambiguously characterized by MALDI-TOF MS and chemical fingerprinting assays. The rate of DNA strand scission at the deoxyribonolactone site was measured in single- and double-stranded DNA under simulated physiological conditions: DNA cleavage occurred with a half-life of approximately 20 h in single-stranded DNA and 32-54 h in duplex DNA, dependent on the identity of the deoxynucleotide paired opposite the lesion site. The initial alpha,beta-elimination reaction was shown to be the rate-determining step for the formation of methylene furanone and phosphorylated DNA products. These investigations demonstrated that the deoxyribonolactone site represents a labile lesion under simulated physiological conditions and forms the basis for further studies of the biological effects of this oxidative DNA damage lesion.

NMR characterization of clustered bistrand abasic site lesions: effect of orientation on their solution structure

A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction of clustered damage: two or more DNA lesions (oxidized bases, abasic sites, or strand breaks) occurring in the same or different strands of the DNA molecule within a single turn of the helix. In spite of arising at a lower frequency than single lesions, clustered DNA damage represents an exotic challenge to the repair systems present in the cells and, in some cases, these lesions may escape detection and/or processing. To understand the structural properties of clustered DNA lesions we have prepared two oligodeoxynucleotide duplexes containing adjacent tetrahydrofuran residues (abasic site analogues), positioned one in each strand of the duplex in a 5′ or 3′ orientation, and determined their solution structure by NMR spectroscopy and molecular dynamics simulations. The NMR data indicate that both duplex structures are right-handed helices of high similarity outside the clustered damage site. The thermal stability of the duplexes is severely reduced by the presence of the abasic residues, especially in a 5′ orientation where the melting temperature is 5°C lower. The structures show remarkable differences at the lesion site where the extrahelical location of the tetrahydrofuran residues in the (AP)2-5′-staggered duplex contrasts with their smooth alignment along the sugar-phosphate backbone in the (AP)2-3′-staggered duplex.

Threading bis-intercalation of a macrocyclic bisacridine at abasic sites in DNA: nuclear magnetic resonance and molecular modeling study.

The macrocyclic bisacridine (CBA) has been reported previously to specifically recognize single-stranded nucleic acid structures, especially DNA hairpins. The binding of the drug with an abasic site-containing oligonucleotide, was investigated by (1)H NMR and molecular modeling. We have used a DNA undecamer, the d(C(1)G(2)C(3)A(4)C(5)X(6)C(7)A(8)C(9)G(10)C(11)) x d(G(12)C(13)G(14)T(15)G(16)T(17)G(18)T(19)G(2)(0)C(21)G(22)) duplex in which the X residue is a stable analogue of the abasic site [3-hydroxy-2-(hydroxymethyl) tetrahydrofuran]. Analysis of the NMR data reveals that the bisacridine molecule forms two different intercalation complexes in a 80/20 (+/- 10) ratio. For the major complex, a molecular modeling study was performed guided by nineteen intermolecular drug-DNA restraints, determined from NOESY spectra. In this model, the ligand interacts in the threading binding mode with an acridine ring intercalated between the C(7)-A(8) and T(15)-G(16) base pairs, while the other acridine ring resides in the abasic pocket. The two linker chains are positioned in the minor and in the major groove, respectively. A comparable study was performed to evaluate the interaction of CBA with the parent unmodified duplex in which X(6) was replaced by an adenine residue. No complex formation was observed when operating in identical conditions. This shows the selective binding of CBA to the abasic site and its potential interest to target the abasic site lesion.

Abasic DNA structure, reactivity, and recognition.

Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiations and alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical point of view,the abasic site is an alkali-labile residue that leads to strand breakage through beta- and delta- elimination. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of synthetic abasic duplexes. Several efficient synthetic methods have thus been developed to introduce the lesion (or a stable analogue) at defined position in the sequence. Physicochemical and spectroscopic examination of such duplexes, including calorimetry, melting temperature, high-field nmr and molecular modeling indicate that the lesion strongly destabilizes the duplex, although remaining in the canonical B-form with structural modifications strictly located at the site of the lesion. Probes have been developed to titrate the damage in DNA in vitro. Series of molecules have been devised to recognize specifically the abasic site, exhibiting a cleavage activity and mimicking the AP nucleases. Others have been prepared that bind strongly to the abasic site and show promise in potentiating the cytotoxic and antitumor activity of the clinically used nitrosourea (bis-chloroethylnitrosurea).

Abasic DNA structure, reactivity, and recognition

Loss of a base in DNA, i.e., creation of an abasic site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously, or under the action of radiations and alkylating agents, or enzymatically as an intermediate in the repair of modified or abnormal bases. The abasic site lesion is mutagenic or lethal if not repaired. From a chemical point of view,the abasic site is an alkali-labile residue that leads to strand breakage through β- and δ- elimination. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of synthetic abasic duplexes. Several efficient synthetic methods have thus been developed to introduce the lesion (or a stable analogue) at defined position in the sequence. Physicochemical and spectroscopic examination of such duplexes, including calorimetry, melting temperature, high-field nmr and molecular modeling indicate that the lesion strongly destabilizes the duplex, although remaining in the canonical B-form with structural modifications strictly located at the site of the lesion. Probes have been developed to titrate the damage in DNA in vitro. Series of molecules have been devised to recognize specifically the abasic site, exhibiting a cleavage activity and mimicking the AP nucleases. Others have been prepared that bind strongly to the abasic site and show promise in potentiating the cytotoxic and antitumor activity of the clinically used nitrosourea (bis-chloroethylnitrosurea). © 2000 John Wiley & Sons, Inc. Biopoly 52: 65–83, 1999

Synthesis of an adenine-pyridinaldoxime-acridine conjugate for recognition of abasic site lesions in DNA

Based on the DNA abasic site targeted artificial enzyme (ATAc 1) previously developed in our laboratory, we designed and prepared a new molecule ( 2) incorporating a nucleophilic oxime function in the linker. A simple convergent strategy was used for the synthesis. The required pyridinic aldehyde derivative 9 was prepared by selenium oxide oxidation of the corresponding methylpyridine, and the two heteroaromatic moieties were successively condensed to afford the conjugate molecule. The oxime function was generated in the last step.