Abasic Lesions Research Articles

Identification and Characterization of Human Apurinic/Apyrimidinic Endonuclease-1 Inhibitors

The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.

Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states.

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as “rollamers”. Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain (“rollamerization”). We demonstrate that such “rollameric” migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor “rollamers” with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic “universal hinge” relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.

8‐Oxoguanine in a quadruplex of the human telomere DNA sequence

8-Oxoguanine is a ubiquitous oxidative base lesion. We report here on the effect of this lesion on the structure and stability of quadruplexes formed by the human telomeric DNA sequence 5'-dG(3)(TTAG(3))(3) in NaCl and KCl. CD, PAGE and absorption-based thermodynamic stability data showed that replacement of any of the tetrad-forming guanines by 8-oxoguanine did not hinder the formation of monomolecular, antiparallel quadruplexes in NaCl. The modified quadruplexes were, however, destabilized in both salts, the extent of this depending on the position of the lesion. These results and the results of previous studies on guanine-to-adenine exchanges and guanine abasic lesions in the same quadruplex show a noticeable trend: it is not the type of the lesion but the position of the modification that determines the effect on the conformation and stability of the quadruplex. The type of lesion only governs the extent of changes, such as of destabilization. Most sensitive sites were found in the middle tetrad of the three-tetrad quadruplex, and the smallest alterations were observed if guanines of the terminal tetrad with the diagonal TTA loop were substituted, although even these substitutions brought about unfavorable enthalpic changes. Interestingly, the majority of these base-modified quadruplexes did not adopt the rearranged folding induced in the unmodified dG(3)(TTAG(3))(3) by potassium ions, an observation that could imply biological relevance of the results.

Abstract C13: A novel DNA damage detection assay for prediction of individual cancer risk

Abstract Background: Unrepaired DNA damage leads to a host of diseases including cancer. Despite the profound impact of DNA damage on human health, little progress has been made in the development of assays capable of accurately measuring induced as well as endogenous in vivo DNA damage. We developed a novel primer-anchored DNA damage detection assay (PADDA) to map and quantify a broad range of DNA lesions at the nucleotide level. Our damage detection assay relies on the principle that the presence of DNA damage on the template can change the polymerase replicative rate, leading to lesion bypass with or without misincorporation, or to lesion-dependent replicative arrest before or opposite the damaged base. Methods: PADDA's specificity, sensitivity and dynamic range were first determined on artificial templates containing engineered DNA lesions and on hydrogen peroxide damaged DNA. To determine the sensitivity of PADDA to detect different spectra of in vivo DNA lesions, we mapped and quantified endogenous and induced DNA damage in wild-type (wt) and repair defective yeast and mice cells. In vivo spontaneous DNA lesions were mapped within the CAN1 gene of S. cerevisiae wt and base excision repair (BER) defective cells and compared to the location of CAN1 published mutations. In vivo induced DNA lesions were mapped within the TRP53 gene in wt and nucleotide excision repair mutant (Xpc−/−) mice irradiated with ultraviolet B (UVB) light. The location of nucleotide lesions were compared to the published p53 mutational spectrum for identical strains. Results: PADDA screens a specific DNA region for base damage, provides detailed fingerprint analysis of base damage and repair at the singlenucleotide level, and quantifies strand-specific DNA damage on a high-throughput scale. The sensitivity of PADDA to detect and map engineered lesions within synthetic oligonucleotides was 100% for abasic and 8-oxodA lesions, 85% for 8-oxoG lesions, and 67% for thymine glycol lesions. PADDA was also able to quantify in vitro induced DNA damage over a wide range of genotoxic exposures (1 μM to 500 mM hydrogen peroxide). Furthermore, PADDA detected significantly higher levels of endogenous damage in yeast cells in stationary phase than in exponential phase, and in yeast BER defective cells than in isogenic wt cells at any phase of growth. Importantly, PADDA detected levels of endogenous DNA lesions that are consistent with published estimates. When mapping endogenous DNA damage, several clusters of DNA lesions were identified in the CAN1 gene. Fisher's exact test showed that the damaged nucleotides detected by PADDA were associated with those previously reported to be the sites of mutations in CAN1. Similarly, a correlation was found between persistent UVB induced DNA damage in the non-transcribed strand of Xpc-/- mice and a previously reported mutational hotspot. These data demonstrate that PADDA is capable of identifying pre-mutagenic DNA lesions in vivo. Conclusion: PADDA is the first assay capable of accurately mapping and quantifying nucleotide and strand specific endogenous DNA damage. PADDA's unprecedented ability to map sites of persistent in vivo endogenous and induced DNA damage before mutation fixation and to quantify strand specific damage may become a critical tool for assessing cancer risk and prevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C13.

An expedient biocatalytic procedure for abasic site precursors useful in oligonucleotide synthesis

Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly regioselective acylation and deacylation reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These compounds are useful precursors for the preparation of phosphoramidites required for the assembly of oligonucleotides containing the tetrahydrofuran abasic lesions.

An Oxidized Abasic Lesion as an Intramolecular Source of DNA Adducts.

5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is a lesion produced in DNA via a variety of damaging agents. The DOB lesion spontaneously generates cis- and trans-but-2-en-1,4-dial (1) via β-elimination. Cis- and trans-but-2-en-1,4-dial forms exocyclic adducts with nucleosides. We used chemically synthesized DNA containing tritiated DOB incorporated at defined sites to examine the reactivity of cis- and trans-but-2-en-1,4-dial. Although the local DNA sequence does not appear to influence the distribution of nucleoside adducts, we find that DOB generates relatively high yields of cis- and trans-but-2-en-1,4-dial nucleoside adducts that likely are promutagenic.

DNA Synthesis across an Abasic Lesion by Yeast Rev1 DNA Polymerase

Abasic (apurinic/apyrimidinic) sites are among the most abundant DNA lesions in humans, and they present a strong block to replication. They are also highly mutagenic because when replicative DNA polymerases manage to insert a nucleotide opposite the lesion, they prefer to insert an A. Rev1, a member of Y-family DNA polymerases, does not obey the A-rule. This enzyme inserts a C opposite an abasic lesion with much greater catalytic efficiency than an A, G, or T. We present here the structure of yeast Rev1 in ternary complex with DNA containing an abasic lesion and with dCTP as the incoming nucleotide. The structure reveals a mechanism of synthesis across an abasic lesion that differs from that in other polymerases. The lesion is driven to an extrahelical position, and the incorporation of a C is mediated by an arginine (Arg324) that is conserved in all known orthologs of Rev1, including humans. The hydrophobic cavity that normally accommodates the unmodified G is instead filled with water molecules. Since Gs are especially prone to depurination through a spontaneous hydrolysis of the glycosidic bond, the ability of Rev1 to stabilize an abasic lesion in its active site and employ a surrogate arginine to incorporate a C provides a unique means for the “error-free” bypass of this noninstructional lesion.

Rapid DNA–protein cross-linking and strand scission by an abasic site in a nucleosome core particle

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.

Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.

Inhibition of Short Patch and Long Patch Base Excision Repair by an Oxidized Abasic Site

5'-(2-Phosphoryl-1,4-dioxobutane) (DOB) is an oxidized abasic lesion that is produced by a variety of DNA damaging agents, including several antitumor antibiotics. DOB efficiently and irreversibly inhibits DNA polymerase β, an essential base excision repair enzyme in mammalian cells. The generality of this mode of inhibition by DOB is supported by the inactivation of DNA polymerase λ, which may serve as a possible backup for DNA polymerase β during abasic site repair. Protein digests suggest that Lys72 and Lys84, which are present in the lyase active site of DNA polymerase β, are modified by DOB. Monoaldehyde analogues of DOB substantiate the importance of the 1,4-dicarbonyl component of DOB for efficient inactivation of Pol β and the contribution of a freely diffusible electrophile liberated from the inhibitor by the enzyme. Inhibition of DNA polymerase β's lyase function is accompanied by inactivation of its DNA polymerase activity as well, which prevents long patch base excision repair of DOB. Overall, DOB is highly refractory to short patch and long patch base excision repair. Its recalcitrance to succumb to repair suggests that DOB is a significant source of the cytotoxicity of DNA damaging agents that produce it.

Translesion Synthesis across Abasic Lesions by Human B-Family and Y-Family DNA Polymerases α, δ, η, ι, κ, and REV1

Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides containing a synthetic abasic lesion (tetrahydrofuran analogue) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, respectively) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t50bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t50bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.

Irreversible Inhibition of DNA Polymerase β by an Oxidized Abasic Lesion

DNA damage is a source of carcinogenicity and is also the source of the cytotoxicity of gamma-radiolysis and antitumor agents, such as the enediynes. The dioxobutane lesion (DOB) is produced by a variety of DNA-damaging agents, including the aforementioned. Repair of DOB is important for maintaining the integrity of the genome as well as counteracting therapeutic agents that target DNA. We demonstrate that the DOB lesion efficiently and irreversibly inhibits repair by DNA polymerase beta (Pol beta), an integral enzyme in base-excision repair. Irreversible inhibition of Pol beta by DOB suggests that this lesion provides a chemical explanation for the cytotoxicity of drugs that produce it and explains previously unexplained observations in the literature concerning abasic lesions that are not repaired efficiently. Finally, these observations provide the impetus for the design of a new family of inhibitors of Pol beta.

Excision of a Lyase-Resistant Oxidized Abasic Lesion from DNA

The C2'-oxidized abasic lesion (C2-AP) is produced in DNA that is subjected to oxidative stress. The lesion disrupts replication and gives rise to mutations that are dependent upon the identity of the upstream nucleotide. Ape1 incises C2-AP, but the 5'-phosphorylated fragment is not a substrate for the lyase activity of DNA polymerase beta. Excision of the lesion is achieved by strand displacement synthesis in the presence of flap endonuclease during which C2-AP and the 3'-adjacent nucleotide are replaced. The oxidized abasic lesion is also a substrate for the bacterial UvrABC nucleotide excision repair system. These data suggest that the redundant nature of DNA repair systems provides a means for removing a lesion that resists excision by short patch base excision repair.

APOBEC3 proteins mediate the clearance of foreign DNA from human cells

Bacteria evolved restriction endonucleases to prevent interspecies DNA transmission and bacteriophage infection. Here, we show that human cells possess an analogous mechanism. APOBEC3A is induced by interferon following DNA detection, and it deaminates foreign double-stranded DNA cytidines to uridines. These atypical DNA nucleosides are converted by the uracil DNA glycosylase UNG2 to abasic lesions, which lead to foreign DNA degradation. This mechanism is evident in cell lines and primary monocytes, where up to 97% of cytidines in foreign DNA are deaminated. In contrast, cellular genomic DNA appears unaffected. Several other APOBEC3s also restrict foreign gene transfer. Related proteins exist in all vertebrates, indicating that foreign DNA restriction may be a conserved innate immune defense mechanism. The efficiency and fidelity of genetic engineering, gene therapy, and DNA vaccination are likely to be influenced by this anti-DNA defense system.

Kinetic Approaches to Understanding the Mechanisms of Fidelity of the Herpes Simplex Virus Type 1 DNA Polymerase

We discuss how the results of presteady-state and steady-state kinetic analysis of the polymerizing and excision activities of herpes simplex virus type 1 (HSV-1) DNA polymerase have led to a better understanding of the mechanisms controlling fidelity of this important model replication polymerase. Despite a poorer misincorporation frequency compared to other replicative polymerases with intrinsic 3′ to 5′ exonuclease (exo) activity, HSV-1 DNA replication fidelity is enhanced by a high kinetic barrier to extending a primer/template containing a mismatch or abasic lesion and by the dynamic ability of the polymerase to switch the primer terminus between the exo and polymerizing active sites. The HSV-1 polymerase with a catalytically inactivated exo activity possesses reduced rates of primer switching and fails to support productive replication, suggesting a novel means to target polymerase for replication inhibition.

Effects of forespore-specific overexpression of apurinic/apyrimidinic endonuclease Nfo on the DNA-damage resistance properties of Bacillus subtilis spores

The effects of overexpression of the apurinic/apyrimidinic DNA endonuclease Nfo on wet and dry heat and UV-C (254 nm) resistance of Bacillus subtilis spores with or without alpha/beta-type small, acid-soluble spore proteins (SASP) were determined. Results revealed that overexpression of Nfo > or =50-fold in spores increased the wet heat resistance of exoA nfo B. subtilis spores (termed alpha(-)beta(-)) that lack most alpha/beta-type SASP, but had no effect on these spores' UV-C resistance. Nfo overexpression also increased these spores' dry heat resistance, and to levels slightly greater than that of wild-type spores. These results are consistent: (1) with wet and dry heat (but not UV-C) generating abasic sites in alpha(-)beta(-) spore DNA; (2) with dry heat generating some of these lesions in spores that retain alpha/beta-type SASP; and (3) indicate that Nfo can repair these abasic lesions following spore germination.

DNA Interstrand Cross-Link Formation by the 1,4-Dioxobutane Abasic Lesion

The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is produced concomitantly with a single-strand break by a variety of DNA-damaging agents that abstract a hydrogen atom from the C5'-position. Independent generation of the DOB lesion in DNA reveals that it reversibly forms interstrand cross-links (ICLs) selectively with a dA opposite the 3'-adjacent nucleotide. Product studies and the use of monoaldehyde models suggest that ICL formation involves condensation of the dialdehyde with the exocyclic amine. Mechanistic studies and inspection of molecular models indicate that the local DNA environment and proximity of the exocyclic amine determine the selectivity for reaction with dA. Proximity control of the electrophile's reactivity is distinct from that of structurally similar freely diffusing molecules. ICL formation by a DOB lesion that is adjacent to a single-strand break is potentially significant because the product constitutes a "clustered" or "complex" lesion. Clustered lesions can lead to highly deleterious double-strand breaks upon nucleotide excision repair.

Photochemical generation and reaction of 2'-substituted analogues of C4'-oxidized abasic lesion

The C4'-oxidized abasic site (1) is one of the oxidatively damaged DNA lesions induced by antitumor bleomycins. The reactivity of 1 with amine under neutral conditions giving lactam 2 and its structural similarity to unmodified DNA suggested the possibility that ODN containing 1 could react with proteins which interact with DNA at lysine residue. In order to provide nucleic acid analogues with useful lysine modifying reactivity, we planned to study reaction of 2'-substituted analogues of 1, such as 3-5, with amine and their corresponding caged precursors 6-8 were synthesized. Uncaging reaction of 16 containing 6 proceeded efficiently. The reaction of obtained 17 containing 3 with Z-Lys-OH did not afford products corresponding to lactam. However, HPLC analysis of the reaction mixture suggested interaction of 3 with Z-Lys-OH.

DNA repair and DNA triplet repeat expansion: the impact of abasic lesions on triplet repeat DNA energetics.

Enhanced levels of DNA triplet expansion are observed when base excision repair (BER) of oxidative DNA base damage (e.g., 8-oxo-dG) occurs at or near CAG repeat sequences. This observation suggests an interplay between processing mechanisms required for DNA repair and expansion pathways that yield genotypes associated with many neurological/developmental disorders. It has been proposed that DNA expansion involves the transient formation within the triplet repeat domains of non-native slipped DNA structures that are incorrectly processed by the BER machinery of repair during DNA synthesis. We show here that replacement within a triplet repeat bulge loop domain of a guanosine residue by an abasic site, the universal BER intermediate, increases the population of slipped/looped DNA structures relative to the corresponding lesion-free construct. Such abasic lesion-induced energetic enhancement of slipped/looped structures provides a linkage between BER and DNA expansion. We discuss how the BER machinery of repair may be influenced by abasic-induced energetic alterations in the properties of regions proximal to and/or within triplet repeat domains, thereby potentially modulating levels of DNA expansion.