Michael G. Debije

DNA Responds to Ionizing Radiation as an Insulator, Not as a "Molecular Wire" **

The yields of radicals trapped on DNA, measured by EPR spectroscopy of oligodeoxyribonucleotide crystals (the EPR spectrum of a single crystal of d(CCCTAGGG) is shown), are found to be very high (0.7 µmol J(-1)) and insensitive to long-range (>10(6) base pairs) versus short-range stacking (8 base pairs) of the bases. These observations are evidence that DNA at 4 K has the properties of an insulator and argue against DNA acting as a molecular wire.

Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release?

Abstract Razskazovskiy, Y., Debije, M. G. and Bernhard, W. A. Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release? The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 μmol J–1. The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient.

On the Efficiency of Hole and Electron Transfer from the Hydration Layer to DNA: An EPR Study of Crystalline DNA X-Irradiated at 4 K

Abstract Debije, M. G., Strickler, M. T. and Bernhard, W. A. On the Efficiency of Hole and Electron Transfer from the Hydration Layer to DNA: An EPR Study of Crystalline DNA X-Irradiated at 4 K. The aim of this project was to gain an improved understanding of how the efficiency of hole and electron transfer from the solvation layer to DNA decreases as a function of distance from DNA. The packing of DNA in crystals of known structure makes it possible to calculate the degree of DNA hydration with a precision that is significantly greater than that achievable for amorphous samples. Previous work on oligodeoxynucleotide crystals has demonstrated that the efficiency of free radical trapping by DNA exposed to ionizing radiation at 4 K is relatively insensitive to base sequence, conformation, counterion, or base stacking continuity. Having eliminated these confounding variables, it is now possible to ascertain the degree of radical transfer that occurs from ionized water as a function of DNA hydration (Γ, in mol water/mol nucleotide). EPR is used to measure the hydroxyl radical concentration in crystals irradiated at 4 K. From a lack of hydroxyl radicals trapped in the inner hydration mantle, we determine that hole transfer to DNA is complete for water molecules located within 8 Å. This corresponds to Γ = 9−11 and indicates that hole transfer is 100% (as efficient as direct ionization of DNA) for water molecules adjacent to DNA. Beyond ∼8 Å (Γ > 10), hydroxyl radicals are observed; thus deprotonation of the water radical cation is seen to compete with hole transfer to DNA as soon as one water intervenes between the ionized water and DNA. The boundary for 0% hole transfer is projected to occur somewhere between 15 and 20 waters per nucleotide. Electron transfer, on the other hand, is 100% efficient across the entire range studied, 4.2 ≤ Γ ≤ 15.6.

Strand Breaks Produced in X-irradiated Crystalline DNA: Influence of Base Sequence

Abstract Razskazovskiy, Y., Debije, M. G. and Bernhard, W. A. Strand Breaks Produced in X-Irradiated Crystalline DNA: Influence of Base Sequence. Radiat. Res. 159, 663–669 (2003). This study reports the radiation-chemical yields for DNA single-strand breaks (SSBs) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes induced by direct ionization using X rays. The DNA fragmentation products, consisting of 3′- and 5′-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single-strand breaks in I and II are 0.16 ± 0.03 μmol/J and 0.07 ± 0.02 μmol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single-strand breaks in DNA and that its action is relatively indiscriminate with respect to base sequence.

Free radical yields in crystalline DNA x-irradiated at 4 K

The objective of this work is to determine the extent to which various structural factors influence the yield of trapped free radicals, G(tfr), in DNA irradiated at 4 K. G(tfr) was measured in a series of 13 different oligodeoxynucleotides using electron paramagnetic resonance (EPR) spectroscopy. Each sample consisted of crystalline duplex DNA for which the crystal structure was verified to be that reported in the literature. We find that the G(tfr) of these samples is remarkably high, ranging from 0.55 to 0.75 micromol/J. The standard deviation in G(tfr) for a given crystal structure is generally small, typically less than +/-10%. Furthermore, G(tfr) does not correlate with DNA base sequence, conformation, counterion or length of base stacking. Two observations point to the importance of DNA packing: (1) The radical yields in crystalline DNA are greater than those determined previously for DNA films (0.2 to 0.5 micromol/J); and (2) the variability in G(tfr) is less in DNA crystals than in DNA films. We conclude that closely packed DNA maximizes radical trapping by minimizing the interhelical solvent space. Furthermore, the high efficiency of electron and hole trapping at 4 K is not consistent with DNA possessing properties of a metallic conductor. Indeed, it behaves as an insulator, whether it is in A-, B-, or Z-form and whether base stacking is short- (8 bp) or long-range (>1000 bp).

Electron Paramagnetic Resonance Evidence for a C3′ Sugar Radical in Crystalline d(CTCTCGAGAG) X-Irradiated at 4 K

Abstract Debije, M. G. and Bernhard, W. A. Electron Paramagnetic Resonance Evidence for a C3′ Sugar Radical in Crystalline d(CTCTCGAGAG) X-Irradiated at 4 K. Radiat. Res. 155, 687–692 (2001). A neutral sugar radical formed by the net loss of hydrogen from C3′ has been identified in crystalline DNA X-irradiated at 4 K. Crystals of duplex d(CTCTCGAGAG), known to be of B conformation, were studied using electron paramagnetic resonance (EPR) spectroscopy. The C3′ radical was identified by using information from dose saturation, power saturation, thermal annealing, and spectrum simulation. The yield of the C3′ radical, G(C3′), is 0.03 ± 0.01 μmol/J, and its concentration does not appear to saturate up to at least 100 kGy. In the region in which total radical concentration increases linearly with dose, the C3′ radical makes up about 4.5% of the total radical population trapped in the oligodeoxynucleotide crystal at 4 K. Based on free base release measured in other oligodeoxynucleotides, we suggest that in d(CTCTCGAGAG) the C3′ radical is responsible for about one-third of the strand breakage events.

Strand breaks in x-irradiated crystalline DNA : alternating CG-oligomers

Abstract Razskazovskiy, Y., Debije, M. G., Howerton, S. B., Williams, L. D. and Bernhard, W. A. Strand Breaks in X-Irradiated Crystalline DNA: Alternating CG Oligomers. Radiat. Res. 160, 334–339 (2003). Direct ionization of crystalline d(CGCGCGCG) and d(CGCGCGCGCG) oligomers produces 3′- and 5′-phosphate-terminated fragments as the main strand breakage products detectable by ion-exchange chromatography. The nature of the base has no effect on the probability of strand breakage at the given site. The yields of 3′-phosphates are systematically lower than the yields of the 5′-phosphates originating from the same cleavage site, pointing to the possible presence of unidentified products with sugar remnants attached to the 3′-end. These results show that direct ionization is efficient at producing single-strand breaks in DNA and its action is relatively indiscriminate with respect to base sequence.

Reductive damage in directly ionized DNA: saturation of the C5=C6 bond of cytosine in d(CGCG)(2) crystals.

Abstract Debije, M. G., Close, D. M. and Bernhard, W. A. Reductive Damage in Directly Ionized DNA: Saturation of the C5u200a=u200aC6 Bond of Cytosine in d(CGCG)2 Crystals. Radiat. Res. 157, 235u200a–u200a242 (2002). Electron paramagnetic resonance (EPR) was used to study an oligodeoxynucleotide duplex of d(CGCG)2 that is known to crystallize in Z-form. After X irradiation at 4 K, EPR data were collected on single crystals and polycrystalline samples as a function of annealing temperature and dose. A radical produced by the net gain of a hydrogen atom at C6 and a proton at N3, Cyt(C6+H, N3+H+)+u200a·, is identified. This radical had not been positively identified in polymeric DNA previously. The Cyt(C6+H, N3+H+)+u200a· makes up about 4u200a% of the total radical population at 4 K, increasing to about 10u200a–u200a15u200a% after the DNA is annealed to 240 K. There appears to be neither an increase nor a decrease in the absolute concentration of Cyt(C6+H, N3+H+)+u200a· upon annealing from 4 K to 240 K. Additionally, the presence of another radical, one due to the net gain of hydrogen at C5 of cytosine, the Cyt(C5+H)·, is implicated. Together, these two radicals appear to account for 60u200a–u200a80u200a% of the reduced species in DNA that has been irradiated at 4 K and annealed to 240 K.