Unlike oxidative processes, radiation produces temporally and spatially grouped ionizations that form molecular radicals in the medium with which it interacts. This unique mechanism of energy deposition leads to spatially clustered lesions (multiply damaged sites (MDS)) and was first proposed by Ward (45
). MDS have been predicted, and for some cases demonstrated (47
), to occur in numerous forms including; base damage in one or both DNA strands, combinations of base damage and single-strand breaks (SSB), structurally simple DSBs, and complex DSBs in which various combinations of these lesions occur proximal to the break (8
). Most of the cytotoxic effects attributed to radiation have been postulated to be due to radiation-induced DSBs, with the greater per lesion cytotoxicity of high-LET radiation being ascribed to the higher likelihood of lesion clustering in close proximity to the DSBs. Such DSBs are believed to be more refractory to repair than simple double-stranded discontinuities (1
). Our recent observations support the notion that non-DSB γ-radiation-induced damage (i.e. base damage and SSBs) upstream from a restriction enzyme induced DSB is a potent inhibitor of human non-homologous end joining (NHEJ) in vitro
, but not of simple direct ligation (11
). Thus a clear understanding of the biochemical mechanisms responsible for radiation cytotoxicity and/or DSB repair cannot be achieved without a clear understanding of the structure of the lesion with which the DSB repair machinery must interact.
125I-TFO targeting has allowed us to obtain DNA containing authentic radiation-induced DSBs within a known sequence context and perform molecular analysis of DSB-associated structural features. In a biological sense, the DSBs produced by 125I decay mimic those produced by high-LET beam irradiation, implying similar structural characteristics. However, unlike beam radiation the 125I decay process results in essentially three modes of DNA damage induction. These are, the direct and indirect low-LET radiation effects caused by the, on average, ~21 Auger electrons emitted by each decay, in conjunction with the non-radiation effects resulting from the charge transfers required from neighboring atoms to neutralize the 125Te daughter atom’s net average +21 charge. All of these decay effects are highly temporally and spatially localized, and thus lead to very localized DNA lesions in the form of DSBs and nucleotide damage proximal to the DSB ends in the duplex DNA’s 125I-TFO-target sequence. Consequently, in addition to providing valuable insights into the radiologic mechanisms and effects of 125I decay in DNA, this system my also be a good model for establishing general categories of structural features that are representative of complex radiation-induced DSBs.
A number of characteristics of these DSBs that do not directly involve damage proximal to the DSB ends have been described in detail elsewhere (24
). Since the substrates used here are the same as those used previously, the general observations concerning the properties of the backbone discontinuities that comprise the DSB are also the same and will not be reiterated here.
The “indirect” and direct effects (including non-radiation effects) of radiation produced by 125
I decay were differentiated by irradiating samples in the presence or absence of the free radical scavenger DMSO. Our observations show that DSB yield (and AP site yield) was not affected by the presence or absence of DMSO, suggesting that most of the strand scission events are the result of direct effects and charge neutralization (24
). These results are consistent with those reported previously by others (25
). On the other hand, the yield of base damage in close proximity to the DSB end was shown to be strongly affected by the presence or absence of DMSO during irradiation, with significantly more base damage being produced by irradiation in the absence of DMSO. Since irradiation occurs in the frozen state, this apparent “indirect effect” probably reflects damage caused by water radicals derived from the first hydration layer of the DNA rather than damage created by interaction of emitted electrons and/or electron stripping via charge neutralization, directly in atoms and bonds of the target DNA. Thus this apparent DMSO scavenging effect may permit some differentiation between damage caused by direct energy deposition (and/or electron abstraction by charge neutralization) in the DNA, and damage that results from secondary reactions with water radicals that are formed within the DNA’s first hydration layer. As such, although some differentiation between modes of damage induction may be identifiable, based on current definitions, DNA damage in this irradiation system is primarily produced by direct effects.
Base damage proximal to the 125
I-induced DSB end of the BglII restriction fragments was assessed by differential enzymatic probing using endo III and Fpg to identify pyrimidine- or purine-derived lesions, respectively. Although these enzymes are known to be capable of some crossover with regard to substrate specificity, kinetic and structure/function analysis of endo III and Fpg indicate that they will preferentially recognize their respective pyrimidine- or purine-derived substrates when presented with DNA containing a mixture of base lesions, particularly in irradiated DNA (50
As expected from its polypurine sequence, the upper strand exhibited only limited sensitivity to endo III cleavage, and only at those fragments closest to the DSB maximum (Fig. and ). The relative increase in endo III sensitivity of the 5′-end labeled upper strand fragments in the (−) DMSO sample with respect to those obtained in the (+) DMSO sample (), suggests a role for scavengeable free radicals in the formation of endo III-recognizable base lesions in the (−) DMSO samples (). In contrast, although the breakage pattern differs, the fragment subset characterized by 3′-end labeling of the upper strand (DSB damaged end) seems to display essentially equal overall sensitivity to cleavage by endo III (). This lack of an overall change in endo III sensitivity in the 3′-end labeled upper strand samples suggests that the endo III-recognizable base lesions closest to the DSB end in this fragment subset, may largely be formed by direct energy deposition effects in the DNA and/or non-radiation “hot atom” mediated ionizations. If this is the case, and the effect is extrapolated to the larger pool of fragments represented by 5′-end labeling of the upper strand, we can speculate that the majority of base lesions produced by “indirect effects” (in this system this is most probably damage derived from water radicals produced in the DNA’s first hydration layer) are likely to occur away from the G4
residue targeted to hybridize with the 125
I-dC of the TFO. Such a result would be consistent with the endo III cleavage sensitivity patterns observed in the 5′-end labeled upper strand (), and consistent with the strand break sensitivity patterns observed for decay of 125
I directly incorporated into short synthetic oligonucleotides as described by Lobachevsky and Martin (28
Fpg probing of the upper strands irradiated in the absence of DMSO (in particular the 5′-end labeled upper strands) displayed the greatest enzyme sensitivity observed in this study, with >50% of the total fragment band density being lost after Fpg treatment (). In contrast, Fpg probing of the upper strand fragments obtained from DNA irradiated in the presence of DMSO exhibited limited enzyme sensitivity, albeit more than for endo III in all respective samples. The increase in Fpg cleavage sensitivity displayed by the (−) DMSO sample relative to the (+) DMSO sample indicates a role for scavengeable free radicals in the formation of radiation-induced purine damage in this system (; ). Therefore, during irradiation in the absence of DMSO the mechanism responsible for this effect is likely to involve first hydration layer derived •OH radical addition to form purine C8-OH-adduct radicals, which subsequently undergo oxidation to form 8-OH-purine lesions (57
). In contrast, when DMSO is present •OH radicals are likely to be scavenged. Thus, the mechanism may involve direct ionization either by the radiated Auger electrons, and /or by charge migration and electron stripping of atoms in the DNA duplex nucleobases to form purine radicals. Direct ionization mechanisms such as these, would also form 8-OH-purines upon hydration and subsequent oxidation after thawing of the samples (57
). Furthermore, those fragments that are most sensitive to Fpg cleavage are those representing the highest strand-break frequency, with the DSB maximum (fragment A5
; ) and the two fragments upstream and downstream of this position displaying the greatest Fpg sensitivity. This observation further supports upper-strand base lesion clustering near the DSB end of the fragments.
As with the upper strand, endo III probing of the polypyrimidine lower strand (3′- and 5′-end labeled) reveals base damage, much of which can be attributed to scavengeable free radicals. In the 3′-end labeled (+) DMSO samples, fragments A2
to C10 showed uniform density loss following endo III treatment (), with a net total density loss of ~24% (). In contrast, endo III probing of the lower strand (−) DMSO sample produced a different cleavage distribution pattern, with fragments T4
displaying greater sensitivity to endo III indicating an increased yield of free radical mediated base damage in these fragments compared to the (+) DMSO sample (). A similar DMSO dependent endo III sensitivity response was seen in the fragments comprising the DSB breakage maxima observed by lower strand 5′-end labeling (DSB damaged end; ). Less total density was lost following endo III treatment of the 5′-end labeled lower strand with respect to that observed by 3′-end labeling, indicating detection of a greater range of lesion containing fragments following 3′-end labeling. As observed for the upper strand, the overall loss of fragment band density following endo III probing of the 5′-end labeled lower strand points to base damage clustering near the DSB end (; ). However, although there does appear to be pyrimidine-derived lesions formed by scavengeable free radicals near the lower-strand DSB end, the yield appears to be less than half that of the purine-derived lesions produced in the upper strand. This result is consistent with the known nucleotide ionization sensitivities (G > A > C > T; (44
)), but it may also reflect a difference in the capacity of endo III and Fpg to recognize and cleave their respective substrates in the complex DSBs investigated here.
As discussed previously (24
), in most of our experiments the electrophoretic conditions required to achieve the necessary resolution for the fragments comprising the DSB breakage spectrum resulted in a lower resolution limit of 9 nucleotides. In addition, our control reactions (, lanes 9-11; and data not shown) indicate that the enzyme preparations used in this work lack phosphatase and non-specific nuclease activities, and the damaged plasmids did not exhibit high levels of non-specific background damage due to the irradiation conditions (). However, significant losses in the total band density of the breakage spectrum were observed following enzymatic probing of the DSB-terminated restriction fragments (). Since the enzyme dependent band density loss of the fragments comprising the breakage spectrum peak measured for the experiments depicted in figures - cannot be accounted for by the creation of new bands of shorter length, or by a density shift from longer bands to shorter bands in the spectrum, lesions may exist within 9 bases of the 32
P label at the fragment’s ends. In the case of fragments labeled at the DSB end (upper and lower strands; Fig. & ), this result is not surprising and is consistent with base lesion clustering near the DSB. However, in the case of fragments labeled at the BglII cut end (Fig. & ) this result is somewhat unexpected. Taken together with the data obtained by labeling the fragment’s DSB ends, these results suggest that the DNA contains base lesion clusters near the DSB end that decrease in frequency with distance from the 125
I decay site (as does the breakage frequency), but then increase in frequency again at distances greater than 24 bases upstream from the decay site. A similar enzymatic sensitivity pattern was observed for these substrates when they were probed for AP sites using endo IV (24
). To determine if this interpretation of our data is correct, we sought to establish electrophoretic conditions that would permit concurrent observation of fragments less than 9 nucleotides in length while still maintaining sufficient resolution at the position of the 125
I-decay target sequence to identify the fragments defining the DSB-induction spectrum. Establishing such conditions permits direct observation of enzyme dependent formation of short fragments representing base damage induction at a distance from the 125
I decay site. The results of one such experiment are illustrated in . As can be seen, Fpg treatment of the upper-strand 32
P-5′-end labeled BglII fragment results in the production of fragments that predominately have lengths of 7 nucleotides or less (). This unusual base-damage-distribution pattern might be explained by charge migration from base lesions produced near the DSB ends to the G residue (and bases in its close proximity) that terminate the polypurine sequence of the TFO binding site. As discussed previously (24
), such a speculation does seem to be consistent with the likely charge migration characteristics of the upper strand sequence (58
), and would appear to be supported by our data.
Fig 8 Identification of increasing base damage frequencies at sites greater than 24 nucleotides upstream from the initial 125I decay site. A) Denaturing polyacrylamide gel (20%) showing changes in band densities and formation of short fragments following Fpg (more ...)
Based upon the known limitations of the enzymes used in this study (with respect to cleavage at sites in which two lesions are closely opposed on opposite strands (59
)), the enzymatic probing analysis suggests a conservative estimate of at least 50% of the upper and lower strands of the 125
I-DSB terminated BglII restriction fragments being likely to contain some form of base damage. Furthermore, many of these fragments are likely to contain multiple lesions. The majority of base lesions in these substrates are likely to occur within eight bases of the DSB end, and they are a direct consequence of the decays responsible for forming the DSBs. This conclusion is based on the lack of increased sensitivity to enzymatic probing of the 49 bp internal control restriction fragment isolated from the irradiated plasmids compared to the same fragment isolated from unirradiated plasmid ().
Finally, GC/MS analysis of the DSB terminated BglII restriction fragments allowed us to identify three specific base lesions that exist within this region. These lesions are 8-hydroxyguanine, 8-hydroxyadenine, and 5-hydroxycytosine. The lack of thymine-derived lesions is consistent with the low yield of lesions detected by endo III probing of the lower strand fragments. This finding is further supported by the absence of detectable thymine glycol following 32P post-labeling analysis of the BglII fragments (data not shown).
This work, together with our work on AP site clustering in this system (24
), allows us to begin construction of a model for the structural organization of a complex radiation-induced DSB. Additional studies to quantitatively assess individual lesion yields within this sequence will permit the development of a probability map for the locations of individual base lesions. This approach should allow us to improve the resolution of our model. However, even without this type of additional analysis, the data we have generated can be used to construct synthetic DSB substrates and develop a more refined analysis of the effect DSB structure has upon the biochemical mechanisms involved in DSB repair.