RESUMEN
DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis-repair of DNA double-strand breaks (DSBs). Unfortunately, how ion beam-induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion-irradiated plants were used for whole-genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non-homologous end-joining (c-NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c-NHEJ to the carbon ion-induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion-beam-induced DSBs in seedlings are likely repaired primarily by a polymerase theta-mediated pathway. Polymerase theta-deficient seedlings were more sensitive to ion beams than the c-NHEJ-deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam-induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements.
Asunto(s)
Arabidopsis , Roturas del ADN de Doble Cadena , Reparación del ADN , Plantones , Semillas , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Plantones/efectos de la radiación , Plantones/genética , Semillas/efectos de la radiación , Semillas/genética , Reparación del ADN/genética , Reparación del ADN por Unión de Extremidades , Reordenamiento Génico/efectos de la radiación , ADN de Plantas/genética , MutaciónRESUMEN
SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.
Asunto(s)
Daño del ADN , Radiación Ionizante , ADN/genética , ADN/efectos de la radiación , Roturas del ADN de Doble Cadena , Reparación del ADN , Microscopía de Fuerza AtómicaRESUMEN
A clustered DNA damage site (cluster), in which two or more lesions exist within a few helical turns, is believed to be a key factor determining the fate of a living cell exposed to a DNA damaging agent such as ionizing radiation. However, the structural details of a cluster such as the number of included lesions and their proximity are unknown. Herein, we develop a method to characterize a cluster by fluorescence anisotropy measurements based on Förster resonance energy transfer (homo-FRET). Plasmid DNA (pUC19) was irradiated with 2.0 and 0.52 MeV/u 4He2+, or 0.37 MeV/u 12C5+ ion beams (linear energy transfer: ~ 70, ~ 150, ~ 760 keV/µm, respectively) and 60Co γ-rays as a standard (~ 0.2 keV/µm) in the solid state. The irradiated DNA was labeled with an aminooxyl fluorophore (Alexa Fluor 488) to the aldehyde/ketone moieties such as apurinic/apyrimidinic sites. Homo-FRET analyses provided the apparent base separation values between lesions in a cluster produced by each ion beam track as 21.1, 19.4, and 18.7 base pairs. The production frequency of a cluster increases with increasing linear energy transfer of radiation. Our results demonstrate that homo-FRET analysis has the potential to discover the qualitative and the quantitative differences of the clusters produced not only by a variety of ionizing radiation but also by other DNA damaging agents.
Asunto(s)
Daño del ADN/efectos de la radiación , Polarización de Fluorescencia/métodos , Algoritmos , Análisis por Conglomerados , Transferencia Resonante de Energía de Fluorescencia/métodos , Rayos gamma/efectos adversos , Plásmidos/genética , Plásmidos/efectos de la radiación , Radiación IonizanteRESUMEN
Complex DNA damage, defined as at least two vicinal lesions within 10-20 base pairs (bp), induced after exposure to ionizing radiation, is recognized as fatal damage to human tissue. Due to the difficulty of directly measuring the aggregation of DNA damage at the nano-meter scale, many cluster analyses of inelastic interactions based on Monte Carlo simulation for radiation track structure in liquid water have been conducted to evaluate DNA damage. Meanwhile, the experimental technique to detect complex DNA damage has evolved in recent decades, so both approaches with simulation and experiment get used for investigating complex DNA damage. During this study, we propose a simplified cluster analysis of ionization and electronic excitation events within 10 bp based on track structure for estimating complex DNA damage yields for electron and X-ray irradiations. We then compare the computational results with the experimental complex DNA damage coupled with base damage (BD) measured by enzymatic cleavage and atomic force microscopy (AFM). The computational results agree well with experimental fractions of complex damage yields, i.e., single and double strand breaks (SSBs, DSBs) and complex BD, when the yield ratio of BD/SSB is assumed to be 1.3. Considering the comparison of complex DSB yields, i.e., DSB + BD and DSB + 2BD, between simulation and experimental data, we find that the aggregation degree of the events along electron tracks reflects the complexity of induced DNA damage, showing 43.5% of DSB induced after 70 kVp X-ray irradiation can be classified as a complex form coupled with BD. The present simulation enables us to quantify the type of complex damage which cannot be measured through in vitro experiments and helps us to interpret the experimental detection efficiency for complex BD measured by AFM. This simple model for estimating complex DNA damage yields contributes to the precise understanding of the DNA damage complexity induced after X-ray and electron irradiations.
Asunto(s)
Daño del ADN , Modelos Genéticos , Análisis por Conglomerados , ADN/química , ADN/genética , ADN/efectos de la radiación , Roturas del ADN de Doble Cadena , Microscopía de Fuerza Atómica , Rayos XRESUMEN
We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (robs) decreases as averaged AP density (λAP: number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60Co γ-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that robs-λAP relationships differed significantly between MMS and NCS. At low AP density (λAP < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60Co γ-rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals.
Asunto(s)
Daño del ADN , ADN/efectos de los fármacos , Polarización de Fluorescencia/métodos , Radioisótopos de Cobalto/farmacología , ADN/química , Colorantes Fluorescentes/química , Rayos gamma , Mesilatos/farmacología , Mutágenos , Cinostatina/farmacologíaRESUMEN
Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.
Asunto(s)
Clonación Molecular/métodos , Daño del ADN , Escherichia coli/genética , Plásmidos/genéticaRESUMEN
Clustered DNA damage, when multiple lesions are generated in close proximity, has various biological consequences, including cell death, chromosome aberrations, and mutations. It is generally perceived as a hallmark of ionizing radiation. The enhanced mutagenic potential of lesions within a cluster has been suggested to result, at least in part, from the selection of the strand with the mutagenic lesion as the preferred template strand, and that this process is relevant to the tolerance of persistent single-strand breaks generated during an attempted repair. Using a plasmid-based assay in Escherichia coli, we examined how the strand bias is affected in mutant strains deficient in different DNA polymerase I activities. Our study revealed that the strand-displacement and 5'-flap endonuclease activities are required for this process, while 3'-to-5' exonuclease activity is not. We also found the strand template that the mutagenic lesion was located on, whether lagging or leading, had no effect on this strand bias. Our results imply that an unknown pathway operates to repair/tolerate the single-strand break generated at a bi-stranded clustered damage site, and that there exist different backup pathways, depending on which DNA polymerase I activity is compromised.
Asunto(s)
Roturas del ADN de Cadena Simple , ADN Polimerasa I , Reparación del ADN , Escherichia coli , Escherichia coli/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa I/genética , Daño del ADN , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación , ADN Bacteriano/genética , ADN Bacteriano/metabolismoRESUMEN
Clustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 µg/µL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2-1890 keV/µm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2-200 keV/µm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on radiation quality and 2. greater LET radiation is more likely to produce a cluster and/or to produce a cluster with shorter distances between lesions by direct effects. This FRET-based proximity estimation of DNA lesions will contribute not only to the identification of clusters and their complexity in a whole genome, but also to the study of their repair mechanism by single-molecular level fluorescence microscopy.
Asunto(s)
Daño del ADN , Radiación Ionizante , Espectrometría de Fluorescencia , ADN/genética , ADN/efectos de la radiación , Colorantes Fluorescentes , AguaRESUMEN
We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10-100 µM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50-60% of base lesions and AP-sites were repaired by 10 µM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.
Asunto(s)
Ácido Ascórbico/química , Roturas del ADN de Cadena Simple/efectos de los fármacos , Reparación del ADN , ADN Bacteriano/efectos de la radiación , Rayos gamma , Plásmidos/efectos de la radiación , Antioxidantes/química , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/genética , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Escherichia coli/genética , Conformación de Ácido Nucleico , Plásmidos/efectos de los fármacos , Plásmidos/genética , Protectores contra Radiación/química , Soluciones/metabolismo , Agua/metabolismoRESUMEN
We have developed a methodology for estimating localization of lesions on double-stranded DNA using fluorescence resonance energy transfer (FRET). We focused on apurinic/apyrimidinic (AP) sites, which are typical DNA lesions induced by radiation and chemicals and produced spontaneously under physiological conditions. Donor-acceptor fluorescent probes with O-amino groups (Alexa Fluor 350-Alexa Fluor 488 dye pair) were used for selectively labeling AP sites. pUC19 plasmid subjected to heat treatment (pH 5.2, 70 °C) was used as a model double-stranded DNA containing AP sites. The results of both FRET analysis and theoretical study enabled us to prove that AP sites induced by the heat treatment are distributed almost randomly along the DNA molecule. This methodology will be useful for estimating the risk of ionizing radiation and chemicals (e.g., pollutants and anticancer agents) based on the probability of producing "clustered DNA damage sites," which are considered to be less easily repairable and, therefore, more harmful to living systems.
Asunto(s)
Daño del ADN , ADN/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Plásmidos/química , ADN/análisis , Plásmidos/análisisRESUMEN
We examined the biological consequences of bi-stranded clustered damage sites, consisting of a combination of DNA lesions, such as a 1-nucleotide gap (GAP), an apurinic/apyrimidinic (AP) site, and an 8-oxo-7,8-dihydroguanine (8-oxoG), using a bacterial plasmid-based assay. Following transformation with the plasmid containing bi-stranded clustered damage sites into the wild type strain of Escherichia coli, transformation frequencies were significantly lower for the bi-stranded clustered GAP/AP lesions (separated by 1bp) than for either a single GAP or a single AP site. When the two lesions were separated by 10-20bp, the transformation efficiencies were comparable with those of the single lesions. This recovery of transformation efficiency for separated lesions requires DNA polymerase I (Pol I) activity. Analogously, the mutation frequency was found to depend on the distance separating lesions in a bi-stranded cluster containing a GAP and an 8-oxoG, and Pol I was found to play an important role in minimising mutations induced as a result of clustered lesions. The mutagenic potential of 8-oxoG within the bi-stranded lesions does not depend on whether it is situated on the leading or lagging strand. These results indicate that the biological consequences of clustered DNA damage strongly depend on the extent of repair of the strand breaks as well as the DNA polymerase in lesion-avoidance pathways during replication.
Asunto(s)
Daño del ADN/genética , ADN Polimerasa I/fisiología , Reparación del ADN/fisiología , Disparidad de Par Base/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Guanina/farmacología , Mutagénesis/fisiología , Organismos Modificados Genéticamente , Eliminación de Secuencia/fisiologíaRESUMEN
Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster 'protects' against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break.
Asunto(s)
Daño del ADN , Mutagénesis , Timina/análogos & derivados , 8-Hidroxi-2'-Desoxicoguanosina , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Escherichia coli/genética , Timina/química , Transformación Bacteriana , Uracilo/químicaRESUMEN
To investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to gamma-radiation and hydroxyurea. This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr. The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1. RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons. The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region. Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast two-hybrid system, suggesting that the proteins form a homodimer. Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after gamma-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage. Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP.
Asunto(s)
Arabidopsis/genética , Arabidopsis/efectos de la radiación , Daño del ADN , ADN de Plantas/genética , Mutación , Rayos Ultravioleta , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromosomas de las Plantas , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Multimerización de ProteínaRESUMEN
Silybin (SLB) and similar analogues, namely, hesperetin (HESP), naringenin (NAN) and naringin (NAR), are believed to be active constituents of natural flavonoids that have been reported as chemopreventive agents for certain cancers. Moreover, SLB and analogues have been determined to fast repair DNA bases from oxidative damage by pulse radiolysis techniques. The present study was designed to evaluate the protective effects of SLB and analogues on soft X-ray-induced damage to plasmid DNA in vitro. The DNA damage was determined by agarose gel electrophoresis. SLB and analogues were found to protect DNA from radiation damage at micromolar concentrations. Among the compounds tested, HESP and SLB were the most effective in preventing X-ray-induced formation of DNA single-strand breaks (SSB). A comparison of these results with other experiments showed that the ability of SLB and analogues to inhibit DNA damage in vitro correlated with the ability of the compounds to scavenge free radicals. Our work revealed that natural flavonoids, SLB and analogues may be used as potent radioprotectors against radiation damage.
Asunto(s)
Daño del ADN , Flavanonas/farmacología , Hesperidina/farmacología , Plásmidos/efectos de los fármacos , Silimarina/farmacología , Antioxidantes/química , Antioxidantes/farmacología , ADN/química , ADN/genética , Roturas del ADN de Cadena Simple/efectos de los fármacos , Roturas del ADN de Cadena Simple/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Electroforesis en Gel de Agar , Flavanonas/química , Hesperidina/química , Modelos Químicos , Estructura Molecular , Plásmidos/genética , Plásmidos/efectos de la radiación , Silibina , Silimarina/químicaRESUMEN
The damaging potential of ionizing radiation arises largely from the generation of clustered DNA damage sites within cells. Previous studies using synthetic DNA lesions have demonstrated that models of clustered DNA damage exhibit enhanced mutagenic potential of the comprising lesions. However, little is known regarding the processes that lead to mutations in these sites, apart from the fact that base excision repair of lesions within the cluster is compromised. Unique features of the mutation frequencies within bi-stranded clusters have led researchers to speculate that the strand containing the mutagenic lesion is preferentially used as the template for DNA synthesis. To gain further insights into the processing of clustered DNA damage sites, we used a plasmid-based assay in E. coli cells. Our findings revealed that the strand containing a mutagenic lesion within a bi-stranded clustered DNA damage site is frequently used as the template. This suggests the presence of an, as yet unknown, strand synthesis process that is unrelated to base excision repair, and that this process plays an important role in mutagenesis. The length of the region of strand preference was found to be determined by DNA polymerase I.
Asunto(s)
Daño del ADN/genética , ADN/biosíntesis , ADN/genética , Roturas del ADN de Doble Cadena/efectos de la radiación , Roturas del ADN de Cadena Simple/efectos de la radiación , ADN Polimerasa I/genética , Reparación del ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Guanina , Mutagénesis/genética , Mutágenos/efectos adversos , Mutación/genética , Tasa de Mutación , Radiación IonizanteRESUMEN
To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70-/- and AtLig4-/-). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70-/- mutant was slightly more sensitive than the AtLig4-/- mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (≥2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11-30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3-4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.
Asunto(s)
Arabidopsis/genética , Arabidopsis/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Rayos gamma , Mutación/genética , Emparejamiento Base/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Mutación INDEL/genética , Tasa de Mutación , Semillas/efectos de la radiación , Eliminación de Secuencia/genética , Transcripción GenéticaRESUMEN
Clustered DNA damage induced by a single radiation track is a unique feature of ionizing radiation. Using a plasmid-based assay in Escherichia coli, we previously found significantly higher mutation frequencies for bistranded clusters containing 7,8-dihydro-8-oxoguanine (8-oxoG) and 5,6-dihydrothymine (DHT) than for either a single 8-oxoG or a single DHT in wild type and in glycosylase-deficient strains of E. coli. This indicates that the removal of an 8-oxoG from a clustered damage site is most likely retarded compared to the removal of a single 8-oxoG. To gain further insights into the processing of bistranded base lesions, several potential repair intermediates following 8-oxoG removal were assessed. Clusters, such as DHT+apurinic/apyrimidinic (AP) and DHT+GAP have relatively low mutation frequencies, whereas clusters, such as AP+AP or GAP+AP, significantly reduce the number of transformed colonies, most probably through formation of a lethal double strand break (DSB). Bistranded AP sites placed 3' to each other with various interlesion distances also blocked replication. These results suggest that bistranded base lesions, i.e., single base lesions on each strand, but not clusters containing only AP sites and strand breaks, are repaired in a coordinated manner so that the formation of DSBs is avoided. We propose that, when either base lesion is initially excised from a bistranded base damage site, the remaining base lesion will only rarely be converted into an AP site or a single strand break in vivo.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Escherichia coli/genética , ADN-Formamidopirimidina Glicosilasa/genética , ADN-Formamidopirimidina Glicosilasa/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Mutación/genética , Timina/análogos & derivados , Timina/metabolismoRESUMEN
After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. The DNA lesions include single strand breaks (SSB), base lesions, sugar damage, and apurinic/apyrimidinic sites (AP sites). Clustered DNA damage, which is defined as two or more of such lesions within one to two helical turns of DNA induced by a single radiation track, is considered to be a unique feature of ionizing radiation. A double strand break (DSB) is a type of clustered DNA damage, in which single strand breaks are formed on opposite strands in close proximity. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints, such as cell death, loss of genetic material, chromosome aberration. Although non-DSB clustered DNA damage has received less attention, there is growing evidence of its biological significance. This review focuses on the current understanding of (1) the yield of non-DSB clustered damage induced by ionizing radiation (2) the processing, and (3) biological consequences of non-DSB clustered DNA damage.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Reparación del ADN/efectos de la radiación , ADN/química , ADN/fisiología , Modelos Biológicos , Animales , ADN/efectos de la radiación , Daño del ADN/efectos de la radiación , Humanos , Radiación IonizanteRESUMEN
PURPOSE: To reveal the reaction process in DNA by Auger electrons using the observed single strand breaks (SSB) and base lesions induced by monochromatic ultrasoft X-rays in dry plasmid DNA film. Desorbed ions from DNA and 2-deoxy-D-ribose thin films were also measured to ascertain the decomposition site in DNA. MATERIALS AND METHODS: Dry plasmid DNA (pUC18) films were irradiated with synchrotron monochromatic ultrasoft X-rays (USX). Two photon energies, 270, and 560 eV, were chosen for the irradiation experiments. Irradiated plasmid DNA was analyzed by agarose gel electrophoresis. The yield of base lesions was determined by the post-irradiation-treatment of the DNA with enzymatic probes (formamidpyrimidine DNA glycosylase [Fpg] and endonuclease III [Nth]). Desorbed ions induced by 540 eV USX irradiation from calf thymus DNA and 2-deoxy-D-ribose thin films were detected by quadrupole-mass spectrometer. RESULTS: Yield of strand breaks and base lesions were obtained by 270 and 560 eV photon energies, respectively. Each yield showed characteristic of the photon energy spectrum. The characteristics of the desorbed ion mass spectra from 2-deoxy-D-ribose and DNA films were strikingly similar with each other. CONCLUSIONS: In this paper we report, for the first time, the yields of base lesions and SSB induced by monochromatic USX. The yield of SSB induced by core-ionization of carbon, nitrogen, and oxygen was two times more frequent than that of valence-electrons. From the comparison of desorbed ion mass spectra of 2-deoxy-D-ribose with DNA films we predict these breaks are likely to be induced by the decomposition of the sugar sites in DNA backbone.