Enhancement of X-ray-induced breaks in DNA bound to molecules containing platinum: a possible application to hadrontherapy.

Complexes made of DNA and chloroterpyridine platinum (PtTC) bound to plasmid DNA were placed in aqueous solution and irradiated with monochromatic X rays tuned to the resonant photoabsorption energy of the L(III) shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) induced by irradiation on a supercoiled DNA plasmid was measured by the production of the circular-nicked and linear forms. To distinguish the contribution of the direct effects of ionization from the indirect effects due to a free radical attack, experiments were also performed in the presence of a hydroxyl free radical scavenger, dimethyl sulfoxide (DMSO). An enhancement of the number of SSBs and DSBs was observed when the plasmids contained the platinum intercalating molecules. A quantitative analysis was made to evaluate the respective contributions of the direct effects (Auger effect) and the indirect effects (free radical attack) to the number of DNA strand breaks. Even when off-resonant X rays were used, the strand break efficiency remained higher than expected based upon the absorption cross section, suggesting that the platinum bound to DNA might be increasing the yield of strand breaks. A mechanism is suggested that involves photoelectrons generated from the ionization of water which efficiently ionize platinum atoms. If this mechanism is correct, then heavy atoms, with a large cross section for ionization by electrons that are bound to the DNA, should behave as a radiosensitizer. This observation may provide insight into understanding the effects of new radiotherapy protocols, related chemotherapeutic agents such as cisplatin, and conventional radiotherapy for the treatment of tumors. A possible way to deliver the dose selectively in a well-defined volume, which uses the properties of the linear energy transfer of atomic ions interacting with matter, is suggested.     

Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons

The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose-response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand-break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons.     

Tris对高LET碳重离子辐照下质粒DNA链断裂的保护作用

利用100 keV/μm碳离子束(初始能量为290 MeV/u)照射溶解于纯水、10 mmol/L Tris、1 mmol/L EDTA及TE 缓冲液中的pUC19质粒DNA.通过琼脂糖凝胶电泳技术分析了不同溶液中各种形态DNA分子所占份额,并计算得到不同剂量下平均每个质粒分子中单链断裂(SSB)及双链断裂(DSB)的数目.发现Tris通过抑制SSB和DSB的产生对碳重离子辐照下的质粒DNA有明显的保护作用,而EDTA能够加剧SSB的产生而抑制DSB的形成.     

Activation-induced cytidine deaminase-dependent DNA breaks in class switch recombination occur during G1 phase of the cell cycle and depend upon mismatch repair

Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in Smu mainly in the G(1) phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G(1) phase cells express AID, UNG, and mismatch repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/SmuTR(-/-) B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously.     

On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays

The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.     

The semi-quantitative comparison of oxidative stress mediated DNA single and double strand breaks using terminal deoxynucleotidyl transferase mediated end labeling combined with a slot blot technique

The accumulation of DNA damages by environmental stresses is represented by the steady state level of single strand breaks (SSBs) and double strand breaks (DSBs). Terminal deoxynucleotidyl transferase (TdT) mediated end labeling is suitable in detecting DSBs, but is unsuitable for SSBs due to its catalyzing characteristics. However, the sensitivity of TdT to detect SSBs may be significantly improved by first denaturing the double strands and expose all the DNA nicks as potential substrates for TdT. By coupling DNA denaturation to slot blot southern hybridization, the authors demonstrate the sensitive detection of SSBs as well as DSBs in 20 ng DNA samples derived from a retinal pigment epithelial cell line treated with tert-butyl hydroperoxide. The signal intensity of denatured and TdT-treated DNA in slot blot hybridization correlated to the amount of SSBs calculated in an S1 nuclease digestion assay. The signal ratio between denatured and non-denatured DNA likely approximates the SSBs/DSBs ratio in genomic DNA. The combination of DNA denaturing, TdT treatment and slot blot hybridization could be a useful method to assess oxidative stress-induced DNA strand damages.     

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.     

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells.

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.     

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.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

^99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, ^99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a ^99mTc-labeled HYNIC-DAPI compound with that of ^99mTc pertechnetate (^99mTcO₄ ⁻). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by ^99mTc-HYNIC-DAPI (1.03) was twice that caused by ^99mTcO₄ ⁻ (0.51), and the number of DSBs increased fivefold in the ^99mTc-HYNIC-DAPI-treated sample compared with the ^99mTcO₄ ⁻ treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the ^99mTcO₄ ^– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the ^99mTc-HYNIC-DAPI-treated samples. These results indicated that ^99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the ^99mTc-labeled compound with DNA. In contrast to these results, ^99mTcO₄ ⁻ induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of ^99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.     

Cluster effects within the local effect model

The local effect model predicts the relative biological effectiveness (RBE) for different ions and cell lines starting from the corresponding experimental photon data and an amorphous track structure model. Here we present an extension of the model that takes cluster effects of single-strand breaks (SSBs) at the nanometer scale into account. In line with the main idea of the local effect model, we take the yields of SSBs and double-strand breaks (DSBs) from experimental photon data and use a Monte Carlo method to distribute them onto the DNA. We score clusters of SSBs where individual SSBs are separated by less than 25 bp as additional DSBs. Assuming that the number of DSBs is a measure of cell lethality, we derive a modified cell survival curve for photons that takes these cluster effects into account. In combination with an improved radial dose distribution, we find that the extended local effect model including cluster effects reproduces most experimental data better than the original local effect model and thus enhances the accuracy of the local effect model.     

Surface shapes and surrounding environment analysis of single‐ and double‐stranded DNA‐binding proteins in protein‐DNA interface

Protein‐DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double‐stranded DNA‐binding proteins (DSBs) and single‐stranded DNA‐binding proteins (SSBs) in protein‐DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single‐stranded DNA) or dsDNA (double‐stranded DNA). Proteins 2016; 84:979–989. © 2016 Wiley Periodicals, Inc.     

Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.     

Discrepancy between the initial DNA damage and cell survival after camptothecin treatment in two murine lymphoma L5178Y sublines

Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID₅₀) after 48 h of incubation were 0·54μM for LY-R cells and 1·25 μM for LY-S cells. Initial numbers of DNA–protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5·6 Gray-equivalents) than in LY-S cells (3·1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT.^1,2 Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4·2 Gray-equivalent SSBs and 5·8 Gray equivalent DSBs) than in LY-S cells (8·0 Gray-equivalent SSBs and 12·0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells.³ Release from G₂ block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G₂, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.     

Numerical Analysis of Etoposide Induced DNA Breaks

Background

Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.

Methodology/Principal Findings

To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.

Conclusions/Significance

These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.     

Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease

DNA double-strand breaks (DSBs) are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs) with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS) can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP) sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2)-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD) in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.