An in vitro DNA double-strand break repair assay based on end-joining of defined duplex oligonucleotides.

DNA double-strand breaks (DSBs) are caused by endogenous cellular processes such as oxidative metabolism, or by exogenous events like exposure to ionizing radiation or other genotoxic agents. Repair of these DSBs is essential for the maintenance of cellular genomic integrity. In human cells, and cells of other higher eukaryotes, DSBs are primarily repaired by the nonhomologous end-joining (NHEJ) DSB repair pathway. Most in vitro assays that have been designed to measure NHEJ activity employ linear plasmid DNA as end-joining substrates, and such assays have made significant contributions to our understanding of the biochemical mechanisms of NHEJ. Here we describe an in vitro end-joining assay employing linear oligonucleotides that has distinct advantages over plasmid-based assays for the study of structure-function relationships between the proteins of the NHEJ pathway and synthetic DNA end-joining substrates possessing predetermined DSB configurations and chemistries.

Radiation-generated short DNA fragments may perturb non-homologous end-joining and induce genomic instability.

Cells exposed to densely ionizing radiation (high-LET) experience more severe biological damage than do cells exposed to sparsely ionizing radiation (low-LET). The prevailing hypothesis is that high-LET radiations induce DNA double strand-breaks (DSB) that are more complex and clustered, and are thereby more challenging to repair. Here, we present experimental data obtained by atomic force microscopy imaging, DNA-dependent protein kinase (DNA-PK) activity determination, DNA ligation assays, and genomic studies to suggest that short DNA fragments are important products of radiation-induced DNA lesions, and that the lengths of DNA fragments may be significant in the cellular responses to ionizing radiation. We propose the presence of a subset of short DNA fragments that may affect cell survival and genetic stability following exposure to ionizing radiation, and that the enhanced biological effects of high-LET radiation may be explained, in part, by the production of increased quantities of short DNA fragments.

Base damage immediately upstream from double-strand break ends is a more severe impediment to nonhomologous end joining than blocked 3'-termini.

Radiation-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions that are typically repaired by nonhomologous end joining (NHEJ) in human cells. Our previous work indicated that the highly cytotoxic DSBs formed by (125)I decay possess base damage clustered within 8 to 10 bases of the break and 3'-phosphate (P) and 3'-OH ends. This study examined the effect of such structures on NHEJ in in vitro assays employing either (125)I decay-induced DSB linearized plasmid DNA or structurally defined duplex oligonucleotides. Duplex oligonucleotides that possess either a 3'-P or 3'-phosphoglycolate (PG) or a ligatable 3'-OH end with either an AP site or an 8-oxo-dG 1 nucleotide upstream (-1n) from the 3'-terminus have been examined for reparability. Moderate to severe end-joining inhibition was observed for modified DSB ends or 8-oxo-dG upstream from a 3'-OH end. In contrast, abolition of end joining was observed with duplexes possessing an AP site upstream from a ligatable 3'-OH end or for a lesion combination involving 3'-P plus an upstream 8-oxo-dG. In addition, base mismatches at the -1n position were also strong inhibitors of NHEJ in this system, suggesting that destabilization of the DSB terminus as a result of base loss or improper base pairing may play a role in the inhibitory effects of these structures. Furthermore, we provide data indicating that DSB end joining is likely to occur prior to removal or repair of base lesions proximal to the DSB terminus. Our results show that base damage or base loss near a DSB end may be a severe block to NHEJ and that complex combinations of lesions presented in the context of a DSB may be more inhibitory than the individual lesions alone. In contrast, blocked DSB 3'-ends alone are only modestly inhibitory to NHEJ. Finally, DNA ligase activity is implicated as being responsible for these effects.

Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining.

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PK(cs) to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PK(cs) appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA-PK(cs) protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.

One-electron oxidation of DNA by ionizing radiation: competition between base-to-base hole-transfer and hole-trapping.

The distance of hole migration through DNA determines the degree to which radiation-induced lesions are clustered. It is the degree of clustering that confers to ionizing radiation its high toxicity. The migration distance is governed by a competition between hole transfer and irreversible trapping reactions. An important type of trapping is reactions that lead to the formation of deoxyribose radicals, which are precursors to free base release (fbr). Using HPLC, fbr was measured in X-irradiated films of d(CGCGCGCGCG)(2) and d(CGCGAATTCGCG)(2) as well as three genomic DNAs: M. luteus, calf thymus, and C. perfringens. The level of DNA hydration was varied from Gamma = 2.5 to 22 mol waters/mol nucleotide. The chemical yields of each base, G(base), were measured and used to calculate the modification factor, M(base). This factor compensates for differences in the GC/AT ratio, providing a measure of the degree to which a given base influences its own release. In the DNA oligomers, M(Gua) > M(Cyt), a result ascribed to the previously observed end effect in short oligomers. In the highly polymerized genomic DNA, we found that M(Cyt) > M(Gua) and that M(Thy) is consistently the smallest of the M factors. For these same DNA films, the yields of total DNA trapped radicals, G(tot)(fr), were measured using EPR spectroscopy. The yield of deoxyribose radicals was calculated using G(dRib)(fr) = approximately 0.11 x G(tot)(fr). Comparing G(dRib)(fr) with total fbr, we found that only about half of the fbr is accounted for by deoxyribose radical intermediates. We conclude that for a hole on cytosine, Cyt(*+), base-to-base hole transfer competes with irreversible trapping by the deoxyribose. In the case of a hole on thymine, Thy(*+), base-to-base hole transfer competes with irreversible trapping by methyl deprotonation. Close proximity of Gua protects the deoxyribose of Cyt but sensitizes the deoxyribose of Thy.

Mechanisms of strand break formation in DNA due to the direct effect of ionizing radiation: the dependency of free base release on the length of alternating CG oligodeoxynucleotides.

The question of how NA base sequence influences the yield of DNA strand breaks produced by the direct effect of ionizing radiation was investigated in a series of oligodeoxynucleotides of the form (d(CG)(n))(2) and (d(GC)(n))(2). The yields of free base release from X-irradiated DNA films containing 2.5 waters/nucleotide were measured by HPLC as a function of oligomer length. For (d(CG)(n))(2), the ratio of the Gua yield to Cyt yield, R, was relatively constant at 2.4-2.5 for n = 2-4 and it decreased to 1.2 as n increased from 5 to 10. When Gua was moved to the 5' end, for example going from d(CG)(5) to d(GC)(5), R dropped from 1.9 +/- 0.1 to 1.1 +/- 0.1. These effects are poorly described if the chemistry at the oligomer ends is assumed to be independent of the remainder of the oligomer. A mathematical model incorporating charge transfer through the base stack was derived to explain these effects. In addition, EPR was used to measure the yield of trapped-deoxyribose radicals at 4 K following X-irradiation at 4 K. The yield of free base release was substantially greater, by 50-100 nmol/J, than the yield of trapped-deoxyribose radicals. Therefore, a large fraction of free base release stems from a nonradical intermediate. For this intermediate, a deoxyribose carbocation formed by two one-electron oxidations is proposed. This reaction pathway requires that the hole (electron loss site) transfers through the base stack and, upon encountering a deoxyribose hole, oxidizes that site to form a deoxyribose carbocation. This reaction mechanism provides a consistent way of explaining both the absence of trapped radical intermediates and the unusual dependence of free base release on oligomer length.

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.

Mechanisms of direct radiation damage in DNA, based on a study of the yields of base damage, deoxyribose damage, and trapped radicals in d(GCACGCGTGC)(2).

Dose-response curves were measured for the formation of direct-type DNA products in X-irradiated d(GCACGCGTGC)(2)prepared as dry films and as crystalline powders. Damage to deoxyribose (dRib) was assessed by HPLC measurements of strand break products containing 3' or 5' terminal phosphate and free base release. Base damage was measured using GC/ MS after acid hydrolysis and trimethylsilylation. The yield of trappable radicals was measured at 4 K by EPR of films X-irradiated at 4 K. With exception of those used for EPR, all samples were X-irradiated at room temperature. There was no measurable difference between working under oxygen or under nitrogen. The chemical yields (in units of nmol/J) for trapped radicals, free base release, 8-oxoGua, 8-oxoAde, diHUra and diHThy were G(total)(fr) = 618 +/- 60, G(fbr) = 93 +/- 8, G(8-oxoGua) = 111 +/- 62, G(8-oxoAde) = 4 +/- 3, G(diHUra) = 127 +/- 160, and G(diHThy) = 39 +/- 60, respectively. The yields were determined and the dose-response curves explained by a mechanistic model consisting of three reaction pathways: (1) trappable-radical single-track, (2) trappable-radical multiple-track, and (3) molecular. If the base content is projected from the decamer's GC:AT ratio of 4:1 to a ratio of 1:1, the percentage of the total measured damage (349 nmol/J) would partition as follows: 20 +/- 16% 8-oxoGua, 3 +/- 3% 8-oxoAde, 28 +/- 46% diHThy, 23 +/- 32% diHUra, and 27 +/- 17% dRib damage. With a cautionary note regarding large standard deviations, the projected yield of total damage is higher in CG-rich DNA because C combined with G is more prone to damage than A combined with T, the ratio of base damage to deoxyribose damage is approximately 3:1, the yield of diHUra is comparable to the yield of diHThy, and the yield of 8-oxoAde is not negligible. While the quantity and quality of the data fall short of proving the hypothesized model, the model provides an explanation for the dose-response curves of the more prevalent end products and provides a means of measuring their chemical yields, i.e., their rate of formation at zero dose. Therefore, we believe that this comprehensive analytical approach, combined with the mechanistic model, will prove important in predicting risk due to exposure to low doses and low dose rates of ionizing radiation.

Unaltered free base release from d(CGCGCG)2 produced by the direct effect of ionizing radiation at 4 K and room temperature.

Unaltered free base release in d(CGCGCG)2 exposed to X rays at 4 K or room temperature was measured by HPLC. Samples were prepared either as films hydrated to a level of Gamma = 2.5 mol water/mol nucleotide or as polycrystalline with Gamma approximately 7.5 mol water/mol nucleotide. X irradiation of films at 4 K, followed by annealing to room temperature, resulted in yields for cytosine and guanine of G(Cyt) = 0.036 +/- 0.001 micromol/J and G(Gua) = 0.090 +/- 0.002 micromol/J. Irradiation of films at room temperature gave similar yields. The yields for polycrystalline d(CGCGCG)2 X-irradiated at room temperature were G(Cyt) = 0.035 +/- 0.005 micromol/J and G(Gua) = 0.077 +/- 0.023 micromol/J. The total free base release yield, G(fbr), was 0.124 +/- 0.008 micromol/J for films and 0.112 +/- 0.028 micromol/J for polycrystalline samples. G(fbr) is believed to be a good estimate of total strand break yield. The yields of total free radicals trapped [G(Sigmafr)] by the d(CGCGCG)2 films at 4 K were measured by EPR. The measured value, G(Sigmafr) = 0.450 +/- 0.005 micromol/J, was used to calculate the yield of trappable sugar radicals, giving G(sugar)(fr) = 0.04-0.07 micromol/J. We found that (1) guanine release exceeded cytosine release by more than twofold, (2) G(sugar)(fr) cannot account for more than half of the free base release, and (3) G(fbr), G(Cyt) and G(Gua) were independent of the sample temperature during irradiation. Finding (1) suggests that base and or sequence influences sugar damage, and finding (2) is consistent with our working hypothesis that an important pathway to strand break formation entails two one-electron oxidations at the same sugar site.

Which DNA damage is likely to be relevant in hormetic responses?

Working under the assumption that hormesis is triggered by specific types of DNA damage, this report focuses on the types of damage which form the signature of ionizing radiation. The key attribute of the signature is the clustering of damage, arising from clusters of energy deposition such that more than one site within a 10 base pair segment of DNA has been chemically altered. A brief overview is given on what is currently believed to be the primary components of clustered damage produced by the direct effect. The overview draws primarily on studies that utilize electron paramagnetic resonance to measure free radical intermediates and gel electrophoresis to measure clustered damage in plasmid DNA. Based on this information, the threshold for a radiation induced biological response is calculated.

An investigation into the mechanisms of DNA strand breakage by direct ionization of variably hydrated plasmid DNA.

The mechanisms by which ionizing radiation directly causes strand breaks in DNA were investigated by comparing the chemical yield of DNA-trapped free radicals to the chemical yield of DNA single strand break (ssb) and double strand break (dsb), as a function of hydration (Gamma). Solid-state films of plasmid pUC18, hydrated to 2.5 < Gamma < 22.5 mol, were X-irradiated at 4 K, warmed to room temperature, and dissolved in water. Free radical yields were determined by EPR at 4 K. With use of the same samples, Gel electrophoresis was used to measure the chemical yield of total strand breaks, which includes prompt plus heat labile ssb; G'total(ssb) decreased from 0.092 +/- 0.016 micromol/J at Gamma= 2.5 to 0.066 +/- 0.008 micromol/J at Gamma= 22.5. Most provocative is that at Gamma= 2.5 the yield of total ssb exceeds the yield of trapped deoxyribose radicals: G'total(ssb) - G'sugar(fr) = 0.06 +/- 0.02 micromol/J. Nearly 2/3 of the strand breaks are derived from precursors other than radicals trapped on the deoxyribose moiety. To account for these nonradical precursors, we hypothesize that strand breaks are produced by two one-electron oxidations at a single deoxyribose residue within an ionization cluster.

The role of hydration in the distribution of free radical trapping in directly ionized DNA.

The purpose of this study was to elucidate the role of hydration (Gamma) in the distribution of free radical trapping in directly ionized DNA. Solid-state films of pUC18 (2686 bp) plasmids were hydrated to Gamma in the range 2.5 < or = Gamma < or = 22.5 mol water/mol nucleotide. Free radical yields, G(Sigmafr), measured by EPR at 4 K are seen to increase from 0.28 +/- 0.01 micromol/J at Gamma = 2.5 to 0.63 +/- 0.01 micromol/J at Gamma= 22.5, respectively. Based on a semi-empirical model of the free radical trapping events that follow the initial ionizations of the DNA components, we conclude that two-thirds of the holes formed on the inner solvation shell (Gamma < 10) transfer to the sugar-phosphate backbone. Likewise, of the holes produced by direct ionization of the sugar-phosphate, about one-third are trapped by deprotonation as neutral sugar-phosphate radical species, while the remaining two-thirds are found to transfer to the bases. This analysis provides the best measure to date for the probability of hole transfer (approximately 67%) into the base stack. It can thus be predicted that the distribution of holes formed in fully hydrated DNA at 4 K will be 78% on the bases and 22% on the sugar-phosphate. Adding the radicals due to electron attachment (confined to the pyrimidine bases), the distribution of all trapped radicals will be 89% on the bases and 11% on the sugar-phosphate backbone. This prediction is supported by partitioning results obtained from the high dose-response curves fitted to the two-component model. These results not only add to our understanding of how the holes redistribute after ionization but are also central to predicting the yield and location of strand breaks in DNA exposed to the direct effects of ionizing radiation.

Correlation of free radical yields with strand break yields produced in plasmid DNA by the direct effect of ionizing radiation.

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(Sigmafr), (ii) radicals trapped on the DNA sugar, Gsugar(fr), (iii) prompt single strand breaks, Gprompt(ssb), (iv) total single strand breaks, Gtotal(ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(fr) were measured by EPR applied to films of pEC (10,810 bp) and pUC18 (2686 bp) plasmids hydrated to Gamma = 22 mol of water/nucleotide and X-irradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(fr) = 0.71 +/- 0.02 and 0.61 +/- 0.01 micromol/J, Gtotal(ssb) = 0.09 +/- 0.01 and 0.14 +/- 0.01 micromol/J, G(dsb) = 0.010 +/- 0.001 and 0.006 +/- 0.001 micromol/J, and Gtota)(ssb)/G(dsb) approximately 9 and approximately 20. Surprisingly, Gsugar(fr) approximately 0.06 mumol/J for pUC18 films, less than half of Gtotal(ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.

What is the initial chemical precursor of DNA strand breaks generated by direct-type effects?

The present study tests the hypothesis that the majority of DNA strand breaks produced by direct-type effects are due to sugar free radical precursors and that these radicals are produced by direct ionization of the sugar-phosphate backbone or by hole transfer to the sugar from tightly bound water. Well-defined crystalline DNA samples of d(CGCG)(2), d(CGCACG:GCGTGC), d(GTGCGCAC)(2), and d((GCACGCGTGC)(2) were irradiated at 4 K, and their free radical dose response determined from 0 to 1800 kGy. A model is proposed that effectively describes the dose response curves. It includes the following parameters: the free radical concentration at saturation C(max), the free radical yields G(b) and G(s), and the destruction constants k(b) and k(s). The subscripts b and s refer to base-centered and sugar-centered radicals, respectively. In each of these systems, the free radical concentration exhibits a remarkable resistance to dose saturation up to at least 1500 kGy. As predicted, G(b) > G(s), the G(b)/G(s) ratio varying between 4 and 12. Likewise, k(b) > k(s), the k(b)/k(s) ratio varying between 28 and 81. The lower cross-section for destruction of the sugar-centered radicals is consistent with the expectation that they are relatively radiation resistant. G(b)/G is between 0.81 and 0.92, indicating that at low doses the bases trap out 80-90% of the total free radical population. The remaining 10-20% are located on the sugar. At high dose, a larger fraction of the radicals are trapped on the backbone as seen from the ratio C(mxS)/C(mxB), which ranges from 3.5 to 8. This unusually late onset of dose saturation closely parallels that observed for strand break products in earlier studies. There is, therefore, a good correlation between the dose response profiles of sugar-trapped radicals and strand breaks. These observations strongly support the hypothesis that sugar radicals are precursors to the majority of strand breaks produced by the direct-type effect in DNA.