The mechanisms of radiation-induced double strand breaks (dsb) in DNA are of long standing interest. This is partly because, among the many occurring types of complex lesions, dsb are simplest to detect. More importantly, it is because the high toxicity of ionizing radiation is directly related to the high efficiency with which radiation forms complex lesions. A number of dsb mechanisms have been proposed: (i) single radical1
, (ii) soliton2
, (iii) pair of radical ions 3,4
, (iv) two radicals born within an ionization cluster5
, and (v) dissociative electron capture6
. Of these, evidence supports (iv) or a combination of (iii) and (iv), as the dominant mechanism7,8
. Therefore, dsb formation entails two (or more) lesions spanning both strands and separated by no more than ~ 3 nm (10 base pairs).
When the opposed lesions are both prompt single strand breaks (ssb), a prompt dsb is formed. If an ssb or damaged base is opposite another damaged base, then excision of the damaged base by a repair enzyme creates a dsb9
. In the case of the direct effect, the predominant lesions are formed via the following free radical intermediates: a carbon-centered deoxyribose radical due to the loss of a hydrogen from one of the five carbon sites, a guanine radical cation, a thymine radical anion, and a cytosine radical anion, respectively10,11
. A two-radical dsb mechanism entails the formation of a precursor consisting of two radicals, with one radical on each strand and separated by a distance d () where d is considered to be ~ 10 base pairs (~ 3 nm). If these two closely spaced radicals exist at the same time, they form what is known in electron paramagnetic resonance (EPR) as a radical pair and they are detected by EPR with a sensitivity comparable to that for detecting isolated radicals. The aim of this work is to determine what fraction of dsb, prompt plus enzymatically induced, can be accounted for by radical pairs.
Figure 1 (a) Represents two radicals (•) present on opposite strands (—) of DNA, at a distance d ≤ 3 nm, giving rise to dsb. (b) Represents two radicals present on the same strand of DNA, at a distance d ≤ 3 nm, giving rise to ssb. (more ...)
Radical pairs are characterized by a strong electron-electron coupling, creating a highly anisotropic interaction described by a tensor D
. The magnitude of the coupling, D, depends on the angle θ between the applied magnetic field B
and the vector d
connecting the two radicals and also on the distance between the two radicals. Because D is proportional to 1/d3
, the unpaired electron-electron interaction decreases quickly with distance. When d > ~ 3 nm the radicals no longer give rise to radical-pair spectral features and are referred to as monoradicals. The formation of radical pairs and dsb, therefore, share the same spatial requirements. If a two radical mechanism is the main precursor to dsb, then radical pairs should exist as intermediates. By irradiating DNA at low temperatures, = 77 K, radical intermediates are trapped and evidence of radical pairs are obtainable through EPR.
One can test for radical pair formation by looking for an EPR signal at half field (g ≈ 4.006), where the Δms
= 2 transition occurs (simultaneous flip of both electrons) or by looking at g ≈ 2.003, where the Δms
= 1 transitions occur for both the paired radicals and the monoradicals. The former has the advantage that any signal at 4.006 is almost assuredly due to radical pairs and the latter has the advantage that the radical pair signal is more than an order of magnitude stronger. Symons and coworkers looked for, and found no evidence of, radical pairs in DNA at 77 K12,13
. But for this null result to take on meaning, two quantities are needed. One is the yield of total dsb, Gtotal
(dsb), and the other is the minimum yield of radical pairs needed for detection by EPR. If the latter is greater than the former, then it is possible that all dsb are formed via the two radical mechanism. Here we fill in the missing information by measuring the detection threshold for radical pairs and comparing it with recently measured dsb yields.
After γ-irradiation of pUC18 films at 279–285 K, Yokaya et al14
measured a prompt dsb yield of G(dsb) = 4 ± 1 nmol/J for DNA hydrated to a level, referred to as Γ, of 14.5 mol H2
O/mol nucleotide. After X-irradiation of pUC18 films at 4 K, Purkayastha et al15
found G(dsb) = 4.0 ± 0.6 nmol/J for DNA at Γ = 15. For this comparison, we converted the yield reported by Yokoya et al from one based on a target mass of DNA alone to one based on DNA plus the mass of its solvation shell. Using enzymes to reveal base damage, it was also possible to measure dsb that involved reduced pyrimidines and oxidized purines, which in the Purkayastha work added 1.5 ± 1.3 and 2.1 ± 1.3 nmol/J, respectively, to the dsb yield. Summing over all types of dsb, the total yield for the work of Purkayastha et al, was Gtotal
(dsb) = 7.6 ± 1.9 nmol/J. For the work of Yokoya et al, Gtotal
(dsb) = 18 ± 5 nmol/J. Based on these yields, if dsb are initiated by a two radical intermediate, the yield of radical pairs in DNA, G(rp-DNA), will be about twice Gtotal
(dsb). The factor of two comes from the probability of radical pair formation on one strand, as in , being equal to formation on opposite strands. Thus, only half of the total radical pairs would give rise to dsb. Based on the findings of Gtotal
(dsb) being in the 8–18 nmol/J range, we should expect G(rp-DNA) in the 16–36 nmol/J range. Our aim here is to determine the threshold for radical pair detection, Gmax
(rp-DNA) , and by comparison with Gtotal
(dsb) determine what fraction of the dsb yield can be accounted for by the radical pair yield.
To our knowledge, the yield of radical pairs produced by ionizing radiation has not been reported for any material. But there are materials where the radical pair signal was reported to be relatively strong. One such material is crystalline thymine, as can be seen from Figure 3 of the work by Boon et al 16
. Irradiation of single crystals of thymine at 77 K has been shown to produce two types of radicals, one due to the net loss of a H of the methyl group, Thy(Me-H)•
, and the other due to the net gain of a H at C6, Thy(C6+H)•17
. Furthermore, there are at least two types of radical pairs in these crystals, both types consisting of Thy(Me-H)•
. The more prominent type has the interconnecting vector d
lying in the bc
crystallographic plane and a separation d (measured from C6 of one radical to C6 of the other) of 0.76 nm. The other type has d
lying in the a*b
plane and d of 0.65 nm18
. The maximum coupling, Dmax
, for the two types is 28 mT and 36 mT, respectively. Because of the large coupling D, the Δms
= 1 signal of the radical pair is easily detected even in the presence of the signal due to the monoradicals trapped in the same sample. The radical pair signal and monoradical signal are both centered at ~ g = 2.0025 but, because the monoradical signal has a spectral width of < 14.5 mT, any signal in the outer wings of the spectrum must be due to radical pairs. These properties make crystalline thymine advantageous for quantifying radical pair production in DNA.
The aim of the work reported here is to use radical pair formation in crystalline thymine as a standard against which the following question can be answered. Is the yield of free radical pairs in DNA sufficient to account for the yield of dsb?