Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival

A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1-2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta (polbeta) are thought to be critical elements in the repair of these SSBs and base damages. XRCC1-deficient cells display a radiosensitive phenotype, while proliferating polbeta-deficient cells are not more radiosensitive. We have recently shown that cells deficient in polbeta display increased radiosensitivity when confluent. In addition, cells expressing a dominant negative to polbeta have been found to be radiosensitized. Here we show that repair of radiation-induced lesions is inhibited in extracts with altered polbeta or XRCC1 status, as measured by an in vitro repair assay employing irradiated plasmid DNA. Extracts from XRCC1-deficient cells showed a dramatically reduced capacity to repair ionizing radiation-induced DNA damage. Extracts deficient in polbeta or containing a dominant negative to polbeta also showed reduced repair of radiation-induced SSBs. Irradiated repaired plasmid DNA showed increased incorporation of radioactive nucleotides, indicating use of an alternative long-patch repair pathway. These data show a deficiency in repair of ionizing radiation damage in extracts from cells deficient or altered in polbeta activity, implying that increased radiosensitivity resulted from radiation damage repair deficiencies.     

Low levels of clustered oxidative DNA damage induced at low and high LET irradiation in mammalian cells

DNA double-strand breaks (DSBs) and locally multiply damaged sites (LMDS) induced by ionizing radiation (IR) are considered to be very genotoxic in mammalian cells. LMDS consist of two or more clustered DNA lesions including oxidative damage locally formed within one or two helical turns by single radiation tracks following local energy deposition. They are thought to be frequently induced by IR but not by normal oxidative metabolism. In mammalian cells, LMDS are detected after specific enzymatic treatments transforming these lesions into additional DSBs that can be revealed by pulsed-field gel electrophoresis (PFGE). Here, we studied radiation-induced DSBs and LMDS in Chinese hamster ovary cells (CHO-K1). After addition of the iron chelator deferoxamine (DFO) or the antioxidant glutathione (GSH) to the cell lysis solution, we observed reduced spontaneous DNA fragmentation and a clear dose-dependent increase of radiation-induced DSBs. LMDS induction, however, was close to background levels, independently of dose, dose rate, temperature and radiation quality (low and high LET). Under these experimental conditions, artefactual oxidative DNA damage during cell lysis could not anymore be confounded with LMDS. We thus show that radiation-induced LMDS composed of oxidized purines or pyrimidines are much less frequent than hitherto reported, and suggest that they may be of minor importance in the radiation response than DSBs. We speculate that complex DSBs with oxidized ends may constitute the main part of radiation-induced clustered lesions. However, this needs further studies.     

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

Measurement of oxidatively generated base damage in cellular DNA

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.     

DNA lesions that signal the induction of radioresistance and DNA repair in yeast

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.     

Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation

Low-linear energy transfer (LET) radiation (i.e., γ- and X-rays) induces DNA double-strand breaks (DSBs) that are rapidly repaired (rejoined). In contrast, DNA damage induced by the dense ionizing track of high-atomic number and energy (HZE) particles is slowly repaired or is irreparable. These unrepaired and/or misrepaired DNA lesions may contribute to the observed higher relative biological effectiveness for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in HZE particle irradiated cells compared to those treated with low-LET radiation. The types of DNA lesions induced by HZE particles have been characterized in vitro and usually consist of two or more closely spaced strand breaks, abasic sites, or oxidized bases on opposing strands. It is unclear why these lesions are difficult to repair. In this review, we highlight the potential of a new technology allowing direct visualization of different types of DNA lesions in human cells and document the emerging significance of live-cell imaging for elucidation of the spatio-temporal characterization of complex DNA damage. We focus on the recent insights into the molecular pathways that participate in the repair of HZE particle-induced DSBs. We also discuss recent advances in our understanding of how different end-processing nucleases aid in repair of DSBs with complicated ends generated by HZE particles. Understanding the mechanism underlying the repair of DNA damage induced by HZE particles will have important implications for estimating the risks to human health associated with HZE particle exposure.     

Radiosensitizers as probes of DNA damage and cell killing

Cell killing and other deleterious biological effects of ionizing radiation are the result of chemical changes to critical targets, initiated at the time of exposure. Electron-affinic radiosensitizers act, primarily, by chemically modifying this radiation damage and its consequent biological expression, and such changes can be used to probe the nature of the cellular radiation target. According to a redox hypothesis of radiation modification, the molecular mechanism of electronic-affinic radiosensitization involves an oxidative interaction of the sensitizer with reactive, potentially damaging target radicals, which competes with reductive processes that restore the target to its undamaged state. The effects have been compared of a series of hypoxic cell radiosensitizers on radiation-induced DNA damage and mammalian cell killing, in order to ascertain the nature of the critical radiation target site(s) involved. Sensitizer efficacy is determined by the ability to oxidize the radiation target and is found to increase exponentially with increasing electron affinity. The threshold redox potential, below which no sensitization occurs, corresponds to the oxidation potential of the target bioradical involved, and is characteristic, and useful in identification, of the particular radiation target. Model product analysis studies of DNA base damage, inorganic phosphate release, single-strand breaks and incorporation of radioactively labelled sensitizer into DNA show a correspondence between the electronic-affinic radiosensitization of DNA damage and cell killing. A careful comparison of the radiosensitization of different DNA sites and cell killing indicates that the sugar-phosphate backbone of DNA, not the heterocyclic bases, is the DNA target site which mimics cell killing in its threshold redox potential and overall radiosensitization response. These results suggest that the enhancement by electron-affinic drugs of radiation damage to the DNA backbone (strand breaks) correlates strongly with, and is the most likely cause of, the radiosensitization of hypoxic cell killing.     

Low efficiency of repair of critical DNA damage induced by low doses of radiation

This study provides an analysis of the development of cellular response to the critical DNA damage and the mechanisms for limiting the efficiency of repairing such damages induced by low doses of ionizing radiation exposure. Based on the data of many studies, one can conclude that the majority of damages occurring in the DNA of the cells after exposure to ionizing radiation significantly differ in their chemical nature from the endogenous ones. The most important characteristic of radiation-induced DNA damages is their complexity and clustering. Double strand breaks, interstrand crosslinks or destruction of the replication fork and formation of long single-stranded gaps in DNA are considered to be critical damages for the fate of cells. The occurrence of such lesions in DNA may be a key event in the etiology and the therapy of cancer. The appearance in the cells of the critical DNA damage induces a rapid development of a complex and ramified network of molecular and biochemical reactions which are called the cellular response to DNA damage. Induction of the cellular response to DNA damage involves the activation of the systems of cell cycle checkpoints, DNA repair, changes in the expression of many genes, reconstruction of the chromatin or apoptosis. However, the efficiency of repair of the complex DNA damage in cells after exposure to low doses of radiation remains at low levels. The development of the cell response to DNA damages after exposure to low doses of radiation does not reach the desired result due to a small amount of damage, with the progression of the phase cell cycle being ahead of the processes of DNA repair. This is primarily due to the failure of signalization to activate the checkpoint of the cell cycle for its arrest in the case of a small number of critical DNA lesions. In the absence of the arrest of the phase cell cycle progression, especially during the G2/M transition, the reparation mechanisms fail to completely restore DNA, and cells pass into mitosis with a damaged DNA. It is assumed that another reason for the low efficiency of DNA repair in the cells after exposure to low doses of radiation is the existence of a restricted access for the repair system components to the complex damages at the DNA sites of highly compacted chromatin.     

Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability

Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.     

DNA-dependent protein kinase complex: a multifunctional protein in DNA repair and damage checkpoint

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated upon DNA damage generated by ionizing radiation or UV-irradiation. It is a three-protein complex consisting of a 470-kDa catalytic subunit (DNA-PKcs) and the regulatory DNA binding subunits, Ku heterodimer (Ku70 and Ku80). Mouse and human cells deficient in DNA-PKcs are hypersensitive to ionizing radiation and defective in V(D)J recombination, suggesting a role for the kinase in double-strand break repair and recombination. The Ku heterodimer binds to double-strand DNA breaks produced by either DNA damage or recombination, protects DNA ends from degradation, orients DNA ends for re-ligation, and recruits its catalytic subunit and additional factors necessary for successful end-joining. DNA-PK is also involved in an early stage of damage-induced cell cycle arrest, however, it remains unclear how the enzyme senses DNA damage and transmits signals to downstream gene(s) and proteins.     

Unique and overlapping functions of the Exo1, Mre11 and Pso2 nucleases in DNA repair

The Mre11 and Pso2 nucleases function in homologous recombination and interstrand cross-link (ICL) repair pathways, respectively, while the Exo1 nuclease is involved in homologous recombination and mismatch repair. Characterization of the sensitivity of single, double and triple mutants for these nucleases in Saccharomyces cerevisiae to various DNA damaging agents reveals complex interactions that depend on the type of DNA damage. The pso2 mutant is uniquely sensitive to agents that generate ICLs and mre11-H125N shows the highest sensitivity of the single mutants for ionizing radiation and methyl methane sulfonate. However, elimination of all three nucleases confers higher sensitivity to IR than any of the single or double mutant combinations indicating a high degree of redundancy and versatility in the response to DNA damage. In response to ICL agents, double-strand breaks are still formed in the triple nuclease mutant indicating that none of these nucleases are responsible for unhooking cross-links.     

A possible role of Ku in mediating sequential repair of closely opposed lesions

One of the hallmarks of ionizing radiation exposure is the formation of clustered damage that includes closely opposed lesions. We show that the Ku70/80 complex (Ku) has a role in the repair of closely opposed lesions in DNA. DNA containing a dihydrouracil (DHU) close to an opposing single strand break was used as a model substrate. It was found that Ku has no effect on the enzymatic activity of human endonuclease III when the substrate DNA contains only DHU. However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonuclease III as well as the amount of free double strand breaks induced by the enzyme. The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III-nicking activity by Ku. Furthermore, there was a concomitant increase in the formation of Ku-DNA complexes when endonuclease III was present. Similar results were also observed with Escherichia coli endonuclease III. These results suggest that Ku reduces the formation of endonuclease III-induced free double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex. In doing so, Ku helps to avoid the formation of the intermediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.     

Effect of intercellular contact on DNA conformation, radiation-induced DNA damage, and mutation in Chinese hamster V79 cells

Chinese hamster V79 cells, when grown as small spheroids in suspension culture, are more resistant to killing by ionizing radiation than when grown as monolayers. We have attempted to determine whether this enhanced survival following irradiation is reflected in DNA damage and repair at the structural level (by measuring alkali-induced DNA unwinding rates from strand breaks) and at the functional level (by measuring resistance to forward mutation at the HGPRT locus). For a given dose of radiation, the unwinding of DNA in high salt/weak alkali was less complete for spheroid DNA than for monolayer DNA, and the rate of repair of radiation damage was faster in spheroid DNA. These differential responses were lost 8 hr after separation of spheroids into single cells, coinciding with loss of radioresistance measured by clonogenicity. In addition, spheroid cells showed fewer numbers of induced mutants per Gray, although, for a given level of survival, the mutation frequency for monolayers and spheroids was identical. These results suggest that conformational changes in DNA resulting from cell growth as spheroids might enhance repair of radiation-induced lesions.     

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.     

Dose–response relationships in chemical carcinogenesis: superposition of different mechanisms of action, resulting in linear–nonlinear curves, practical thresholds, J-shapes

The shape of a carcinogen dose–cancer incidence curve is discussed as the result of a superposition of dose–response relationships for various effects of the carcinogen on the process of carcinogenesis. Effects include direct DNA damage, e.g., by covalent binding, indirect DNA damage, e.g., by increased formation of reactive oxygen species or interaction with DNA replication or chromosome integrity. The ‘fixation' of a DNA adduct as a heritable mutation depends on its pro-mutagenic potency and on the rates of DNA repair and DNA replication. Endogenous and unavoidable DNA damage is responsible for a background rate of the process of mutagenesis and carcinogenesis and forms the basis of spontaneous cancer incidence. For DNA-reactive carcinogens, linearity of the dose response at the low-dose end is expected. With increasing dose, saturation of DNA repair can introduce a sublinearity (example: dimethylnitrosamine). Stimulation of cell division as a result of high-dose toxicity and regenerative proliferation also results in a sublinear deviation from low-dose linearity. If the DNA-damaging potency of the carcinogen is low in comparison with the high-dose effects, the linear part of the low dose–cancer incidence curve might be hidden within the background variability. Under such conditions, ‘practical thresholds' could be discussed (formaldehyde). If a carcinogen increases the rate of cell division or the level of oxidative stress at high dose but has an antimitogenic or antioxidative effect at low dose, a J-shaped (or: U-shaped) curve with a decrease of the spontaneous tumor incidence at low dose could result (caffeic acid; TCDD). This phenomenon has been observed even under conditions of a genotoxic contribution (ionizing radiation; diesel exhaust particles). For a mechanism-based assessment of a low-dose cancer risk, information on the various modes of action and modulations should be available over the full dose range, and models should be refined to incorporate the respective information.     

Clustered DNA damages induced by x rays in human cells

Although DNA DSBs are known to be important in producing the damaging effects of ionizing radiation in cells, bistranded clustered DNA damages-two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns-are postulated to be difficult to repair and thus to be critical radiation-induced lesions. Gamma rays can induce clustered damages in DNA in solution, and high-energy iron ions produce DSBs and oxidized pyrimidine clusters in human cells, but it was not known whether sparsely ionizing radiation can produce clustered damages in mammalian cells. We show here that X rays induce abasic clusters, oxidized pyrimidine clusters, and oxidized purine clusters in DNA in human cells. Non-DSB clustered damages comprise about 70% of the complex lesions produced in cells. The relative levels of specific cluster classes depend on the environment of the DNA.     

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.