The yield of strand breaks resulting from direct-type effects in crystalline DNA X-irradiated at 4 K and room temperature.

Radiation damages DNA through two distinct types of reaction pathways, direct-type and indirect-type.1 The indirect-type pathways entail reactions of water radiolysis products with DNA, for example, the formation of strand breaks by hydroxyl radical attack, which has been extensively studied.2 Direct-type effects are caused by direct ionization of DNA or by transfer of holes and electrons to DNA from the surrounding hydration waters.3-5 The direct component may constitute a significant fraction of the total radiation damage to DNA in cells where water radiolysis products are effectively scavenged by the medium.6 Much less is known about the direct-type mechanisms and related quantitative characteristics, especially in regards to strand break formation. It is known that electron attachment to DNA is not a significant source of strand breaks.7 Sugar radicals generated through one-electron oxidation of the sugar–phosphate moiety has been implicated as the main source of strand scission from direct-type damage.7 This study reports the first direct measurement of radiation–chemical yields of individual strand break products induced by the direct effect in DNA. Two DNA oligonucleotides, d(CGCG)2 (1) and d(CGCGCG)2 (2), duplexes in crystalline form, were X-irradiated at two different temperatures, 4 and 293 K. HPLC in combination with photodiode array detection was used to identify and measure the chemical yields of all cleavage fragments that retained at least one unaltered base. Product identifications are based on comparison with the retention times and optical absorption spectra of authentic compounds and are supported by MALDI-TOF measurements.8 Crystals of oligonucleotides offer the advantage of exact knowledge of the helical packing and conformation. Both 19 and 210 are in Z-conformation, and the hydration levels are relatively low (7.1 and 7.2 waters per nucleotide, respectively),4 well below the level required for detectable production of OH radicals from the waters of hydration.5 Previous work has shown that trapped hydroxyl radicals are not detected at 4 K in crystals of either 1 or 2.4 Indirect-type reactions, therefore, cannot be a significant source of strand breaks in these samples. A typical HPLC chromatogram of irradiated 2 is shown in Figure 1 along with the reference and zero dose chromatograms. Chromatograms of the irradiated 1 differ from Figure 1 by the absence of peaks corresponding to end-phosphorylated tetramers and pentamers, and by the parent-compound retention time. From comparison of the crystal and reference spectra, all major damage products found in the crystal are represented in the reference sample and vice versa. The fragmentation products are identified as free bases, 3′-, and 5′-end phosphorylated strand fragments. The production of other degradation products not present in the reference sample is minimal. For example, there is no evidence of 3′-phosphoglycolates that are the products of the H4′-abstraction from DNA in the presence of oxygen (see ref 12 for a review). Figure 1 HPLC profile at 260 nm of X-irradiated11 2 (top), unirradiated 2 (middle), and the reference sample (bottom). The irradiated sample received a dose of 300 kGy at 293 K. Samples were dissolved in a 20 mM phosphate buffer and held at 70 °C for 30 . The standard irradiated crystalline sample is about 200 μg, and the products each make up <1% of this initial sample. Sample sizes for 1 were larger, and the standard deviations were significantly smaller. The yield of each DNA strand fragment is given in Figure 2. Figure 2 Yields (in μmol/J) of products in 1 and 2 after irradiation at (above) and 293 K (below). Yields were determined from the slopes of the least-squares lines fit to the dose response data. The errors, given in parentheses, are the standard deviations . In summary, this study finds that (1) the major products of strand breakage from the direct-type effect are free bases and oligodeoxynucleotides with 3′-, and 5′-phosphate end groups and (2) the yield of immediate strand breaks is between 0.05 and 0.15 μmol/J.