Clustered DNA Damage Sites Research Articles

The Influence of Clustered DNA Damage Containing Iz/Oz and OXOdG on the Charge Transfer through the Double Helix: A Theoretical Study.

The genome-the source of life and platform of evolution-is continuously exposed to harmful factors, both extra- and intra-cellular. Their activity causes different types of DNA damage, with approximately 80 different types of lesions having been identified so far. In this paper, the influence of a clustered DNA damage site containing imidazolone (Iz) or oxazolone (Oz) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG) on the charge transfer through the double helix as well as their electronic properties were investigated. To this end, the structures of oligo-Iz, d[A1Iz2A3OXOG4A5]*d[T5C4T3C2T1], and oligo-Oz, d[A1Oz2A3OXOG4A5]*d[T5C4T3C2T1], were optimized at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the aqueous phase using the ONIOM methodology; all the discussed energies were obtained at the M06-2X/6-31++G** level of theory. The non-equilibrated and equilibrated solvent-solute interactions were taken into consideration. The following results were found: (A) In all the discussed cases, OXOdG showed a higher predisposition to radical cation formation, and B) the excess electron migration toward Iz and Oz was preferred. However, in the case of oligo-Oz, the electron transfer from Oz2 to complementary C4 was noted during vertical to adiabatic anion relaxation, while for oligo-Iz, it was settled exclusively on the Iz2 moiety. The above was reflected in the charge transfer rate constant, vertical/adiabatic ionization potential, and electron affinity energy values, as well as the charge and spin distribution. It can be postulated that imidazolone moiety formation within the CDL ds-oligo structure and its conversion to oxazolone can significantly influence the charge migration process, depending on the C2 carbon hybridization sp2 or sp3. The above can confuse the single DNA damage recognition and removal processes, cause an increase in mutagenesis, and harm the effectiveness of anticancer therapy.

Chromatin and the Cellular Response to Particle Radiation-Induced Oxidative and Clustered DNA Damage

Exposure to environmental ionizing radiation is prevalent, with greatest lifetime doses typically from high Linear Energy Transfer (high-LET) alpha particles via the radioactive decay of radon gas in indoor air. Particle radiation is highly genotoxic, inducing DNA damage including oxidative base lesions and DNA double strand breaks. Due to the ionization density of high-LET radiation, the consequent damage is highly clustered wherein ≥2 distinct DNA lesions occur within 1–2 helical turns of one another. These multiply-damaged sites are difficult for eukaryotic cells to resolve either quickly or accurately, resulting in the persistence of DNA damage and/or the accumulation of mutations at a greater rate per absorbed dose, relative to lower LET radiation types. The proximity of the same and different types of DNA lesions to one another is challenging for DNA repair processes, with diverse pathways often confounding or interplaying with one another in complex ways. In this context, understanding the state of the higher order chromatin compaction and arrangements is essential, as it influences the density of damage produced by high-LET radiation and regulates the recruitment and activity of DNA repair factors. This review will summarize the latest research exploring the processes by which clustered DNA damage sites are induced, detected, and repaired in the context of chromatin.

Fluorescence anisotropy study of radiation-induced DNA damage clustering based on FRET.

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 0.52MeV/u 4He2+, or 0.37MeV/u 12C5+ ion beams (linear energy transfer: ~ 70, ~ 150, ~ 760keV/μm, respectively) and 60Co γ-rays as a standard (~ 0.2keV/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.

Strand with mutagenic lesion is preferentially used as a template in the region of a bi-stranded clustered DNA damage site in Escherichia coli

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.

Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells.

High-linear energy transfer (LET) heavy ions cause higher therapeutic effects than low-LET radiation due to lower dependency on oxygen concentration in tumor cell killing. The lethality after irradiation largely depends on DNA double-strand breaks (DSBs), however the detailed LET dependency for DSB induction under oxic and hypoxic conditions has not been reported. Therefore, we evaluated the oxygen enhancement ratio (OER) of heavy ion-induced DSB induction using a highly-optimized flow cytometry-based method of γ-H2AX detection. Non-small cell lung cancer (NSCLC) A549 cells were exposed to X-ray, carbon-ion and iron-ion radiations under oxic or hypoxic condition. As a DSB marker, the γ-H2AX signal was measured 1 h postirradiation and analyzed by flow cytometry. DSB slope values were calculated as DSB induction per Gy. Our method was able to detect high-LET radiation-induced DSBs even from clustered DNA damage sites. We also showed a decrease in OER value in an LET-dependent manner regardless of radiation type. In summary, we demonstrated a simple, quick and highly-optimized flow cytometry-based method of DSB analysis that detects DSBs induced by heavy-ion radiation for hypoxic and nonhypoxic cancer cells. Our study may provide a useful biological basis for heavy-ion radiotherapy.

Efficiency of radiation-induced base lesion excision and the order of enzymatic treatment

Purpose: To clarify whether initial base excision repair processes at clustered DNA damage sites comprising multiple base lesions affect subsequent excision processes via the formation of additional strand breaks by glycosylase and apurinic/apyrimidinic (AP) endonuclease base excision enzymes.Materials and methods: Plasmid DNA (pUC18) as a model DNA molecule was exposed to high-linear-energy-transfer (LET) ionizing radiation (He2+ or C6+ ions) or low-LET ionizing radiation (X-rays) under various conditions to produce varied radical-scavenging effects. pUC18 was then treated sequentially or simultaneously with two bacterial base excision enzymes (glycosylases), namely, endonuclease III and formamidopyrimidine-DNA glycosylase, which convert pyrimidine (or abasic [AP] site) and purine (or AP site) lesions to single-strand breaks (SSB), respectively. Yields of additional SSB or double-strand breaks (DSB) as digestion products were examined after changing the order of enzymatic treatment.Results: There were few differences among the enzymatic treatments, indicating that treatment order did not affect the final yields of additional SSB or DSB formed by glycosylase activity. This suggests that of the total damage, the fraction of clustered damage sites with a persistent base lesion dependent on the order of glycosylase treatment was insignificant if present.Conclusion: Base lesion clusters induced by high- or low-LET radiation appear three or more base pairs apart, and are promptly converted to a DSB by glycosylase, regardless of the order of enzymatic treatment.

A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch

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.

Efficient cleavage of single and clustered AP site lesions within mono-nucleosome templates by CHO-K1 nuclear extract contrasts with retardation of incision by purified APE1

Clustered DNA damage is a unique characteristic of radiation-induced DNA damage and the formation of these sites poses a serious challenge to the cell’s repair machinery. Within a cell DNA is compacted, with nucleosomes being the first order of higher level structure. However, few data are reported on the efficiency of clustered-lesion processing within nucleosomal DNA templates. Here, we show retardation of cleavage of a single AP site by purified APE1 when contained in nucleosomal DNA, compared to cleavage of an AP site in non-nucleosomal DNA. This retardation seen in nucleosomal DNA was alleviated by incubation with CHO-K1 nuclear extract. When clustered DNA damage sites containing bistranded AP sites were present in nucleosomal DNA, efficient cleavage of the AP sites was observed after treatment with nuclear extract. The resultant DSB formation led to DNA dissociating from the histone core and nucleosomal dispersion. Clustered damaged sites containing bistranded AP site/8-oxoG residues showed no retardation of cleavage of the AP site but retardation of 8-oxoG excision, compared to isolated lesions, thus DSB formation was not seen. An increased understanding of processing of clustered DNA damage in a nucleosomal environment may lead to new strategies to enhance the cytotoxic effects of radiotherapeutics.

Order effect of repair processes to clustered DNA damage

In a living cell, a multiply damaged site in DNA is thought to be processed by several different pathways simultaneously or sequentially. Under this situation, the cellular response to the lesion cluster might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of initial repair protein, affecting the DNA-binding or lesion-excision activities of latter repair proteins. For example, a cluster comprised an AP site or SSB and base lesions is formed after one of the lesions in a base lesion cluster is excised by a glycosylase. It has been predicted that the enzymatic activity of a protein binding to a lesion could be affected by a second lesion located nearby. In the present study, we investigate whether initial base excision repair process affects secondary process on the clustered DNA damage site produced by high linear energy transfer irradiation.Plasmid DNA (pUC18) used as model DNA were irradiated in a solution (10 mM Tris and 1 mM EDTA) with C6+ ions (60 keV/µm) obtained from HIMAC (NIRS, Chiba, Japan) according to our previous method [ 1], or He2+ ions from TIARA (JAEA, Takasaki, Japan) in a fully hydrated film [ 2]. We used two base excision repair enzymes to examine the order effect of BER processes, Nth and Fpg, which convert pyrimidine and purine lesions to an SSB, respectively. After exposure, the sample was incubated with Nth (or Fpg) at 37°C for 30 min. Then, we added another enzyme Fpg (or Nth) and incubate for another 30 min. We also simultaneously treated the DNA sample with both enzymes for 1 h. After the incubation, the samples were analysed by agarose (1%) gel electrophoresis to determine the yield of conformational changes of the plasmid from closed circular form to open or linear form. These changes show productions of an enzymatically induced SSB (the former) or DSB (the latter).A typical example of the dependence of the loss of closed circular form DNA on radiation dose is shown in Fig. 1a for carbon ion irradiation. The reciprocal of the slope of each graph shows yield of prompt SSBs [n(SSB)prompt] for the sample without any enzymatic treatment, or [n(SSB)prompt] + enzymatically induced SSBs [n(SSB)Nth(or Fpg, or Nth+Fpg)]. The SSB yield for the sample treated with Nth first and then Fpg is very slightly less than that of other treatments (<10%). The dose responses of the linear form DNA production are shown in Fig. 1b. There were no significant differences among the enzymatic treatments. These results indicate that the configuration change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme. The most of base lesion clusters produced in the track of carbon ions might be converted to strand breaks, some of which form double-strand breaks (DSBs), by glycosylase activity in living cells. Fig. 1.Dependence of (a) the loss of closed-circular DNA and (b) the number of linear form DNA DSB determined from the fraction of the linear form of DNA, on carbon ion dose. The arrows indicate the order of the enzymatic treatments.

Dependence of the yields of AP sites and AP clusters produced in plasmid DNA on scavenging capacity and LET

An apurinic or apyrimidinic site (AP site) has known to be one of the typical DNA lesions induced by ionizing irradiation to cells. A clustered DNA damage site composed of AP sites (AP clusters) can be visualized as a double-strand break (DSB) by a treatment of DNA sample with endonuclease IV proteins as an enzymatic probe. AP clusters are efficiently induced in human cells by low LET γ- and X-irradiation [ 1] with the similar yields with those for the clusters which contain pyrimidine or purine base lesions. However, there has been very little knowledge of the mechanistic aspects of the production of AP sites and AP clusters. Recently, we reported the dependence of the yields of AP sites and AP clusters, as well as base lesions, strand breaks and base lesion clusters induced by irradiation of carbon ions (LET: 13 and 60 keV/μm) obtained from HIMAC (NIRS, Chiba, Japan) on radical scavenging capacity in the DNA samples [ 2].In the present study, we have performed theoretical calculation for the production of AP sites as well as those of strand breaks and base lesions by the carbon ion exposure (13 keV/μm). A Monte Carlo track structure simulation code for ion tracks, TRACION, was used for this work according to our previous study [ 3]. As a DNA model molecule, simple linear DNA with 150 bp was applied for the calculation. Several scavenging capacities of the samples are tested to estimate the effect of indirect action of diffusible OH radicals in the damage induction in the experimental conditions.We assumed that the AP site formation pathway originates from a common intermediate of an OH radical adduct with that of single-strand breaks (SSBs) or base lesions (Scheme 1). In order to reproduce the experimentally obtained yields of AP sites, we newly determined the branching ratio of induction of AP site to that of SSBs or base lesions to be 17:33 or 2:48, respectively, in the simulation. The calculated yields of AP site, as well as SSB, were well consistent with those for experimentally obtained (Fig. 1). However, the calculated yields for AP clusters were one-fifth of those for experimental data. The yields for DSBs in lower scavenging capacities were also one-fifth of those for experimental ones in a lower scavenging capacity, thought they were well consistent with each other above a cell mimetic condition (>3 × 108 s). In order to bridge a gap between theoretical and experimental yields, we need a novel mechanism of damage-clustering such as dissociative low energy electron attachment or hole-migration onto a DNA molecule. Fig. 1.Dependence of the yield of AP site or AP cluster (A) and prompt SSB or prompt DSB (B) on scavenging capacity in the sample. The experimental data below a scavenging capacity of 109 s are cited from [ 2] and those at 1010 s in (B) are from [ 4]. Scheme 1.Reactions of DNA with an OH radical and the branching ratios among the damage induction pathways.

Effects of (5'S)-5',8-cyclo-2'-deoxyadenosine on the base excision repair of oxidatively generated clustered DNA damage. A biochemical and theoretical study.

The presence of 5',8-cyclo-2'-deoxyadenosine (5'S)-cdA induces modifications in the geometry of the DNA duplex in the 5'-end direction of the strand and in the 3'-end direction of the complementary strand. As a consequence, the enzymes are probably not able to adjust their active sites in this rigid structure. Additionally, clustered DNA damage sites, a signature of ionising radiation, pose a severe challenge to a cell's repair machinery, particularly base excision repair (BER). To date, clusters containing a DNA base lesion, (5'S)-cdA, which is repaired by nucleotide excision repair, have not been explored. We have therefore investigated whether bistranded clusters containing (5'S)-cdA influence the repairability of an opposed AP site lesion, which is repaired by BER. Using synthetic oligonucleotides containing a bistranded cluster with (5'S)-cdA and an AP site at different interlesion separations, we have shown that in the presence of (5'S)-cdA on the 5'-end side, repair of the AP site by the BER machinery is retarded when the AP site is ≤8 bases from the (5'S)-cdA. However, if (5'S)-cdA is located on the 3'-end side with respect to the AP site, the effect on its repair is much weaker and totally disappears for distances ≥8 bases.

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

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

Biological Consequences of Radiation-induced DNA Damage: Relevance to Radiotherapy

DNA damage of exposed tumour tissue leading to cell death is one of the detrimental effects of ionising radiation that is exploited, with beneficial consequences, for radiotherapy. The pattern of the discrete energy depositions during passage of the ionising track of radiation defines the spatial distribution of lesions induced in DNA with a fraction of the DNA damage sites containing clusters of lesions, formed over a few nanometres, against a background of endogenously induced individual lesions. These clustered DNA damage sites, which may be considered as a signature of ionising radiation, underlie the deleterious biological consequences of ionising radiation. The concepts developed rely in part on the fact that ionising radiation creates significant levels of clustered DNA damage, including complex double-strand breaks (DSB), to kill tumour cells as clustered damage sites are difficult to repair. This reduced repairability of clustered DNA damage using specific repair pathways is exploitable in radiotherapy for the treatment of cancer. We discuss some potential strategies to enhance radiosensitivity by targeting the repair pathways of radiation-induced clustered damage and complex DNA DSB, through inhibition of specific proteins that are not required in the repair pathways for endogenous damage. The variety and severity of DNA damage from ionising radiation is also influenced by the tumour microenvironment, being especially sensitive to the oxygen status of the cells. For instance, nitric oxide is known to influence the types of damage induced by radiation under hypoxic conditions. A potential strategy based on bioreductive activation of pro-drugs to release nitric oxide is discussed as an approach to deliver nitric oxide to hypoxic tumours during radiotherapy. The ultimate aim of this review is to stimulate thinking on how knowledge of the complexity of radiation-induced DNA damage may contribute to the development of adjuncts to radiotherapy.

Condensation of DNA—A putative obstruction for repair process in abasic clustered DNA damage

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form.Oligomer DNA duplexes (47bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid–oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer–plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer–plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration.

A methodology for estimating localization of apurinic/apyrimidinic sites in DNA using fluorescence resonance energy transfer

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.

Hierarchy of lesion processing governs the repair, double-strand break formation and mutability of three-lesion clustered DNA damage

Ionising radiation induces clustered DNA damage sites which pose a severe challenge to the cell’s repair machinery, particularly base excision repair. To date, most studies have focussed on two-lesion clusters. We have designed synthetic oligonucleotides to give a variety of three-lesion clusters containing abasic sites and 8-oxo-7, 8-dihydroguanine to investigate if the hierarchy of lesion processing dictates whether the cluster is cytotoxic or mutagenic. Clusters containing two tandem 8-oxoG lesions opposing an AP site showed retardation of repair of the AP site with nuclear extract and an elevated mutation frequency after transformation into wild-type or mutY Escherichia coli. Clusters containing bistranded AP sites with a vicinal 8-oxoG form DSBs with nuclear extract, as confirmed in vivo by transformation into wild-type E. coli. Using ung1 E. coli, we propose that DSBs arise via lesion processing rather than stalled replication in cycling cells. This study provides evidence that it is not only the prompt formation of DSBs that has implications on cell survival but also the conversion of non-DSB clusters into DSBs during processing and attempted repair. The inaccurate repair of such clusters has biological significance due to the ultimate risk of tumourigenesis or as potential cytotoxic lesions in tumour cells.

Induction of DNA Strand Breaks, Base Lesions and Clustered Damage Sites in Hydrated Plasmid DNA Films by Ultrasoft X Rays around the Phosphorus K Edge

To characterize the DNA damage induced by K-shell ionization of phosphorus atom in DNA backbone on the level of hydration, the yields of DNA strand breaks and base lesions arising from the interaction of ultrasoft X rays with energies around the phosphorus K edge were determined using dry and fully hydrated pUC18 plasmid DNA samples. Base lesions and bistranded clustered DNA damage sites were revealed by postirradiation treatment with the base excision repair proteins endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of prompt single-strand breaks (SSBs) with dry DNA irradiated at the phosphorus K resonance energy (2153 eV) is about one-third that below the phosphorus K edge (2147 eV). The yields of prompt double-strand breaks (DSBs) were found to be less dependent on the X-ray energy, with the yields being about two times lower when irradiated at 2153 eV. Heat-labile sites were not produced in detectable amounts. The yields of base lesions were dependent on the energy of the X rays, especially when the DNA was fully hydrated. Bistranded clustered DNA damage sites, revealed enzymatically as additional DSBs, were produced in dry as well as in hydrated DNA with all three energies of X rays. The yields of these enzyme-sensitive sites were also lower when irradiated at the phosphorus K resonance energy. On the other hand, the yields of prompt SSBs and enzyme-sensitive sites for the two off-resonance energies were, larger than those determined previously for gamma radiation. The results indicate that the photoelectric effect caused by X rays and dense ionization and excitation events along the tracks of low-energy secondary electrons are more effective at inducing SSBs and enzyme-sensitive sites. The complex types of damage, prompt and enzymatically induced DSBs, are preferentially induced by phosphorus K resonance at 2153 eV rather than simple SSBs and isolated base lesions, particularly in hydrated conditions. It is concluded that not only the phosphorus K resonance and resulting emission of low-energy LMM-Auger electrons ( approximately 120 eV) but also the level of hydration plays an important role in the induction of complex damage in plasmid DNA.

Extensive Repair of DNA Double-Strand Breaks in Cells Deficient in the DNA-PK-Dependent Pathway of NHEJ after Exclusion of Heat-Labile Sites

Abstract Double-strand breaks (DSBs) can be generated in the DNA of mammalian cells when heat-labile sites induced by ionizing radiation within a clustered DNA damage site are thermally transformed to single-strand breaks (SSBs) and combine with other SSBs or heat-labile sites in the opposite DNA strand. When this thermal transformation of heat-labile sites to SSBs occurs during DNA preparation using high-temperature lysis, the DSB yield is overestimated by including DSBs not present in the tested cell. Low-temperature lysis avoids this artifact but shows slower repair kinetics for prompt DSBs than high-temperature lysis. The apparent slowdown of DSB repair after low-temperature lysis is particularly pronounced in mutants defective in the DNA-PK-dependent pathway of NHEJ (D-NHEJ) and has led to the postulate that these cells may actually be entirely deficient in DSB repair. Since our work as well as that of others provides strong evidence for DSB repair in D-NHEJ-deficient cells by alternative repair pathways operating as a backup (B-NHEJ), we re-examine here DSB repair in several D-NHEJ-deficient mutants using low-temperature lysis. The results demonstrate extensive but slow repair of DSBs after low-temperature lysis indicative of a robust function of B-NHEJ. At the same time, the results demonstrate a pronounced sensitivity of heat-labile sites to lysis at 37 degrees C, which indicates that DSBs generated by transformation of heat-labile sites may be in part a reality that the irradiated cell faces at all times.

Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic

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.

2P-113 一本鎖切断を含むクラスター損傷の分子動力学シミュレーション(核酸・構造物性,第46回日本生物物理学会年会)

2P-113 一本鎖切断を含むクラスター損傷の分子動力学シミュレーション(核酸・構造物性,第46回日本生物物理学会年会)