Base Excision Repair Endonuclease Research Articles

Breakage of cytoplasmic chromosomes by pathological DNA base excision repair.

Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridges5-8. These structures are associated with fragile nuclear envelopes that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by adenine deaminases acting on RNA (ADAR enzymes) to generate deoxyinosine. Deoxyinosine is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, N-methyl-purine DNA glycosylase11,12 (MPG). These abasic sites are cleaved by the BER endonuclease, apurinic/apyrimidinic endonuclease12 (APE1), creating single-stranded DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the deoxyinosine base from the DNA strand of RNA-DNA hybrids, which we demonstrate using purified proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with pre-existing defects such as the DNA base abnormality described here.

Contribution of Base Damages to the Molecular Radiosensitization Mechanism of Platinum Chemotherapeutic Drugs

AbstractThe measurement of clustered damages in Pt‐chemotherapeutic‐drugs‐DNA complexes induced by low‐energy (0‐30 eV) electrons (LEEs) may provide further understanding of the synergy mechanism in cancer treatment with concomitant chemoradiation therapy (CRT). Five‐monolayer films of Pt‐DNA complexes, containing cisplatin, carboplatin and oxaliplatin with ratio 5:1 were irradiated with mono‐energetic electrons of 10 eV, the most prominent energy for bond dissociation by LEE impact. Damages to plasmid DNA including crosslinks, SSBs, DSBs and the loss of the supercoiled configuration were analyzed by the electrophoresis. Base damages (BDs) occurring within two turns of DNA helix were detected by the treatment of E. coli base excision repair endonuclease (Nth and Fpg). The total DNA damages to Pt‐DNA complexes was found to be 350 ± 42, 296 ± 45 and 434 ± 46 ×10−15 electron−1molecule−1, respectively, while the percentages of BDs in this total were 56%, 54% and 60%, respectively. Compared to unmodified DNA, binding of Pt‐drugs to DNA increased BDs by factors of 1.9, 1.6 and 2.6, respectively, which partly resulted in significant enhancements of potentially lethal lesions (i. e., crosslinks, double strand breaks (DSBs) and non‐DSB cluster damages). These results reveal the significant role that LEEs play in the formation of clustered DNA damages related to BDs, which are expected to contribute to the higher efficacy of cancer treatment by CRT with the Pt‐drugs investigated.

Molecular efficacy of radio- and chemotherapy sequences from direct DNA damage measurements

Purpose: To investigate the molecular aspects of the synergy between ionizing radiation and platinum (Pt) chemotherapeutic agents in cancer treatment with chemoradiation therapy (CRT) by measuring damages induced by low-energy electrons (LEE) to DNA bound to cisplatin. LEE are produced abundantly by any type of ionizing radiation and cisplatin represents a typical Pt-chemotherapeutic agents.Materials and methods: Our strategy involves two parallel administrations of cisplatin and irradiation with a 4.6 and 9.6 eV electron fluence of 1.1 × 1012: (1) LEE bombardment of supercoiled DNA and its subsequent reaction with cisplatin; (2) the reaction of DNA with cisplatin followed by LEE irradiation. The damage yields for the loss of supercoiled (LS), single-strand breaks (SSB) and double-strand breaks (DSB) were obtained from gel electrophoresis analysis. Base modifications were revealed by treating the samples with Escherichia coli base excision repair endonuclease (Nth and Fpg).Results: The yields were deduced from the respective time–response for the reaction of DNA with cisplatin. The results show that binding cisplatin to DNA followed by LEE irradiation, consistently yields more DNA damages than the reverse order. In comparison to non-treated DNA, administration (2) results in an increase of LS and SSB of 1.4–3.3 folds and of DSB by more than an order of magnitude. Furthermore, after enzyme treatment, the yields of DSB rise by factors of 5.3–15.4, indicating a large increase of clustered damages, which should at least partially translate into an increase of lethal damages in cancer cells during the CRT.Conclusions: Our results demonstrate that a strong synergy between radiation and cisplatin can only be achieved at the molecular level, if the drug is present at the time of irradiation. Furthermore, this work confirms the LEE mechanism previously proposed to explain the synergy between radiation and Pt drugs in CRT. It involves chemical sensitization of DNA prior to irradiation, to facilitate strand breaks and clustered damages induced by the highly reactive LEE.

Avoidance of APOBEC3B-induced mutation by error-free lesion bypass.

APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce.

Unified Mechanism for the Generation of Isolated and Clustered DNA Damages by a Single Low Energy (5–10 eV) Electron

Clustered DNA damages are the most detrimental modifications induced by ionizing radiation in cells and several mechanisms have been proposed for their formation. We report measurements of such damages induced by a single low energy electron via the formation of the two major core-excited resonances of DNA located at 4.6 and 9.6 eV. Cross-links and single and double strand breaks (SSBs and DSBs) are analyzed by gel electrophoresis. Treatment of irradiated samples with Esherichia coli base excision repair endonucleases reveals base damages (BDs). DSBs resulting from such treatments arise from clustered damages consisting of at least two BDs or one BD accompanied by a SSB. The total DNA damages induced by 4.6 and 9.6 eV electrons are 132 ± 32 and 201 ± 36 × 10–15 electron–1 molecule–1, comprising 43% and 52% BDs, respectively. We propose a unifying mechanism to account for these clustered damages, DSBs, and single BDs, as well as all previously measured isolated lesions.

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function.

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N 2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.

NPM1 and APE1: nucleolar teamwork in controlling base excision DNA repair

Apurinic/apyrimidinic endonuclease 1 (APE1), the main abasic endonuclease in base excision repair (BER) acting on oxidative and alkylation DNA lesions, is a promising target for anticancer therapy. In nucleoli, APE1 interacts through N‐terminal lysine residues with nucleophosmin 1 (NPM1). We used a reconstitution strategy to address the impact of the NPM1‐APE1 interaction on resistance to genotoxic damage. Lysine‐mutated APE1 proteins were expressed while endogenous APE1 was silenced by siRNA to address the role of APE1 acetylation during cell response to DNA damage and a NPM1 knock‐out system to determine its role in modulating APE1 in BER pathway. We demonstrated that APE1 acetylation at lysines 27, 31, 32 and 35, enhanced by genotoxic stress, inhibited binding to NPM1 and promoted re‐localization of APE1 from nucleoli to the nucleoplasm. Interestingly, NPM1 stimulated APE1 BER activity in vivo, since cells lacking NPM1 were more sensitive to bleomycin or alkylating agents and displayed impaired DNA repair. These results highlight an unexpected role for NPM1 and the nucleolus in coordinating APE1 and BER pathways, and point to acetylation at critical lysine residues for controlling subnuclear protein trafficking during DNA repair.This work is supported by grants from AIRC and FIRB.

Clustered DNA Damage in Irradiated Human Diploid Fibroblasts: Influence of Chromatin Organization

Clustered DNA damages are induced by ionizing radiation and are defined as two or more lesions within one or two helical turns. The aim of this study was to investigate the induction and repair of clustered DNA damage in cells with emphasis on the influence of structural differences in the chromatin organization. Human fibroblasts were irradiated with X rays and induced DSBs and clustered damages were quantified using pulsed-field gel electrophoresis combined with postirradiation incubation with the base excision repair endonuclease Fpg, which recognizes oxidized purines and cleaves the strand at sites inducing strand breaks. Hence clustered damages appear in enzyme-treated samples as additional DSBs. The chromatin was modified by different pretreatments that resulted in structures with varying compactness and levels of free radical scavenging capacity. We found that the induction of DSBs and clustered damages increased linearly with dose in all structures and that both types of lesions were allocated randomly within the nucleus. The induction yields increased with decreasing compactness of chromatin, and the chromatin effect was larger for clustered lesions than for DSBs. Clustered damages were processed efficiently with a fast and a slow repair component similar to that for induced DSBs.

The SET Complex Acts as a Barrier to Autointegration of HIV-1

Retroviruses and retrotransposons are vulnerable to a suicidal pathway known as autointegration, which occurs when the 3′-ends of the reverse transcript are activated by integrase and then attack sites within the viral DNA. Retroelements have diverse strategies for suppressing autointegration, but how HIV-1 protects itself from autointegration is not well-understood. Here we show that knocking down any of the components of the SET complex, an endoplasmic reticulum-associated complex that contains 3 DNases (the base excision repair endonuclease APE1, 5′-3′ exonuclease TREX1, and endonuclease NM23-H1), inhibits HIV-1 and HIV-2/SIV, but not MLV or ASV, infection. Inhibition occurs at a step in the viral life cycle after reverse transcription but before chromosomal integration. Antibodies to SET complex proteins capture HIV-1 DNA in the cytoplasm, suggesting a direct interaction between the SET complex and the HIV preintegration complex. Cloning of HIV integration sites in cells with knocked down SET complex components revealed an increase in autointegration, which was verified using a novel semi-quantitative nested PCR assay to detect autointegrants. When SET complex proteins are knocked down, autointegration increases 2–3–fold and chromosomal integration correspondingly decreases ∼3-fold. Therefore, the SET complex facilitates HIV-1 infection by preventing suicidal autointegration.

Oxidative stress alters base excision repair pathway and increases apoptotic response in apurinic/apyrimidinic endonuclease 1/redox factor-1 haploinsufficient mice

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-κB, and the major 5′-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 ( Apex +/−) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-κB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex +/− mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex +/− mice exposed to 2-NP displayed a significant decline in 3′-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER.

Repair of oxidative guanine damage in plasmid DNA by indoles involves proton transfer between complementary bases.

We have used the single electron oxidizing agent (SCN)(2)(*)(-) (generated by gamma-irradiation of aqueous thiocyanate) to produce guanyl radicals in plasmid DNA. The stable product(s) formed from these radicals can be detected after conversion with a base excision repair endonuclease to single strand breaks. The yield of enzyme-induced breaks is decreased by the presence during irradiation of indole compounds. Rate constants for the reduction of DNA guanyl radicals by these indoles can be calculated from the concentration dependence of the attenuation in the yield of enzyme sensitive sites. Indoles bearing electron-donating groups (methoxy or methyl) appear to react at the diffusion-controlled rate, but those bearing electron-withdrawing groups (cyano or nitro) are significantly less reactive. At physiological pH values, the reduction of a DNA guanyl radical involves the transfer of a proton as well as an electron. Comparison of the kinetic results with literature thermodynamic data suggests that the source of this proton is the complementary base-paired cytosine.

Repair of guanyl radicals in plasmid DNA by electron transfer is coupled to proton transfer.

By using gamma-irradiation in the presence of thiocyanate ions, we have generated guanyl radicals in plasmid DNA. These can be detected by using an Escherichia coli base excision repair endonuclease to convert their stable end products to strand breaks. The yield of enzyme-sensitive sites is strongly attenuated by the presence of micromolar concentrations of one of a series of singly substituted phenols, and it is possible to derive bimolecular rate constants for the reduction of DNA guanyl radicals by these phenols. More strongly reducing phenols were found to react more rapidly. This electron-transfer reaction also involves a proton transfer. By comparing the expected energetics of the reaction with the observed rate constants, the electron transfer is found to be mechanistically coupled with the proton transfer.

Modulation of the 3′→5′-Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5′-incised Abasic DNA Product

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.

Modification of ionizing radiation clustered damage: estimate of the migration distance of holes through DNA via guanyl radicals under physiological conditions

Purpose : Guanyl radicals are produced in DNA when it is subjected to oxidation or ionizing radiation. The sites at which stable products can be identified can be located dozens of base pairs away from the initial site of the electron loss. This migration will modify the spatial distribution of damage and tends to mitigate the clustering of initial damage generally associated with ionizing radiation. The migration distance is presumably a function of the lifetime of the intermediate guanyl radical, and we wished to quantify the relationship between them. Materials and methods : Aqueous solutions containing plasmid DNA and thiocyanate ions were treated with γ-irradiation. These conditions result in the very efficient production of guanyl radicals in the plasmid. We quantified the formation of stable guanine oxidation products in the plasmid as strand breaks by using the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The effect of two additives on the yield of guanine oxidation, nitrite ions and the DNA binding ligand doxorubicin (adriamycin), were examined. Results : The presence during irradiation of the DNA-binding ligand doxorubicin attenuated the yields of stable oxidized guanine products formed. The additional presence of nitrite decreased this effect of doxorubicin. Conclusion : Because doxorubicin binds strongly to DNA, its ability to attenuate guanine oxidation can be interpreted in terms of the migration distance of the intermediate guanyl radical. Because nitrite repairs these intermediate guanyl radicals by electron transfer, its presence during irradiation decreases their lifetime. Therefore, we derived an estimate of the migration distance of guanyl radicals as a function of their lifetime. The presence in cells of antioxidants such as glutathione sets an upper limit to the likely lifetime and, therefore, the migration distance of guanyl radicals. It was concluded that the migration of guanyl radicals may not decrease the clustering of DNA damage in vivo to a great extent.

One-electron oxidation of plasmid DNA by selenium(V) species

Purpose : To employ the γ-radiation-generated selenium(V) one-electron-oxidizing agent SeO 3 •- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. Materials and methods : Plasmid DNA in aerobic aqueous solution was irradiated with 137 Cs γ-rays (662 keV). The solutions also contained up to 4 ×10 -2 mol dm -3 sodium selenate (Na 2 SeO 4) and/or up to 10 -1 mol dm -3 sodium biselenite (NaHSeO 3) , as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. Results : In general, γ-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after γ-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after γ-irradiation in the presence of 10 -2 mol dm -3 sodium selenate and 10 -1 mol dm -3 glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO 3 •-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. Conclusion : By comparing the yields of breaks produced after γ-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.

Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength

Purpose : Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN) 2 £ -. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN) 2 £ - is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of oneelectron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. Materials and methods : Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137 Cs n -irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. Results : The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). Conclusions : It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN) 2 £ - and DNA-G £ +. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G £ + /DNA of E 7 = +1.39 - 0.01V.

Reaction of guanyl radicals in plasmid DNA with biological reductants: chemical repair of DNA damage produced by the direct effect of ionizing radiation

Purpose : It has been previously argued that the use of the one-electron oxidants (SCN) 2 •- and Br 2 •- with plasmid DNA leads to the formation of DNA guanyl radicals. These guanyl radical species are intermediates in the DNA damage produced by processes such as photo-ionization and ionizing irradiation. The present paper evaluates the use of thallium(II) ions (Tl II OH +) as the one-electron oxidant, and also determines rate constants for the reduction (repair) of guanyl radicals in plasmid DNA by a variety of reducing agents including the biologically important compounds ascorbate and glutathione. Materials and methods : Aqueous solutions of plasmid DNA containing 10 -3 mol dm -3 thiocyanate or thallous ions and a reducing agent (azide, nitrite, ferrocyanide, hexachloroiridate(III), iodide, ascorbate, glutathione, glutathione disulphide, methionine, tyrosine, 5-hydroxyindole-3-acetic acid, 10 -7 -10 -4 mol dm -3) were irradiated with 137 Cs γ-rays (662 keV). After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). Strand break yields after incubation were quantified by means of agarose gel electrophoresis. Results : High yields of FPG-sensitive sites produced by the oxidants (SCN)2 •- and Tl II OH + were strongly attenuated by the presence of the reducing agents. Conclusions : From the results, it is possible to arrive at estimates of the rate constants for the reduction of the DNA guanyl radical by the reducing agents. Values lie in the range 10 4 -10 7 dm 3 mol -1 s -1. Using the values for ascorbate and glutathione, it is possible to estimate an upper limit on the order of milliseconds for the lifetime of DNA guanyl radicals under cellular conditions. The implication is that there may well be a significant chemical repair of DNA base damage by the direct effect of ionizing radiation.

Redox reactivity of guanyl radicals in plasmid DNA

Purpose : It has been previously argued that γ-irradiation of plasmid DNA in the presence of thiocyanate ions produces products recognized by the E. coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG), and there that derive from an intermediate guanyl radical species. The wish was to characterize the reactivity of this intermediate with reducing agents. Materials and methods : Aqueous solutions of plasmid DNA containing either bromide or thiocyanate (10 -3 to 10 -1 mol dm -3) and also one of six other additives (azide, ferrocyanide, iodide, nitrite, promethazine, tryptophan, 10 -7 to 10 -3 mol dm -3) were subjected to 137 Cs γ-irradiation (662 keV). After irradiation, the plasmid was incubated with FPG. Strand break yields before and after incubation were determined by agarose gel electrophoresis under neutral conditions. Results : The very high yields of FPG-sensitive sites in the presence of SCN - or Br - decreased significantly with increasing concentrations of all of the six additives, with promethazine and tryptophan being the most efficient additives, and azide and iodide the least. Conclusions : From the results it is possible to estimate values of the rate constants for the reduction of the DNA guanyl radical (5x10 5, 2x10 5, 10 7 and 10 7 dm 3 mol -1 s -1 for ferrocyanide, nitrite, promethazine and tryptophan respectively).

The Inositol Polyphosphate 5-Phosphatases and the Apurinic/Apyrimidinic Base Excision Repair Endonucleases Share a Common Mechanism for Catalysis

Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.

Mechanism of DNA damage by thiocyanate radicals

Purpose : It was previously shown that γ-irradiation of aqueous solutions of plasmid DNA in the presence of millimolar concentrations of thiocyanate ions leads to the formation in very high yields of sites recognized by the base excision repair endonuclease formamido-pyrimidine-DNA N -glycosylase (FPG). The authors wished to characterize the mechanism responsible for the production of these FPG-sensitive sites. Materials and methods : An aqueous solution of plasmid DNA containing thiocyanate ions was irradiated with 137 Cs γ-rays. After irradiation, aliquots were treated with FPG. Break yields were determined using neutral agarose gel electrophoresis. Results : The yield of FPG-sensitive sites decreased with decreasing enzyme activity, increasing thiocyanate concentration, increasing dose-rate, increasing ionic strength, increasing nitrite or iodide concentration, and decreasing oxygen concentration. Conclusions: The observations suggest that the monomeric thiocyanate radical SCN •is an intermediate in the reaction, and that the yields of FPG-sensitive sites are determined by competition between the disproportionation of the dimeric radical anion (SCN) 2 •- and the fate of a one-electron oxidized guanine species in DNA. The latter can react with oxygen to produce an FPGsensitive site or can be reduced without producing an FPGsensitive site. The results help to clarify the mechanisms responsible for DNA damage by the direct eÚect of ionizing radiation.