The effects of ionizing radiation on DNA have been studied for decades as DNA is the critical cellular target responsible for cell killing. Ionizing radiation produces a wide variety of lesions within DNA, such as base modifications, base-free sites, single-strand and double-strand breaks and DNA–protein crosslinks (1
). These lesions, if unrepaired, may produce detrimental biological consequences, such as mutagenesis, carcinogenesis and even cell death. Consequently, the accurate identification and measurement of these DNA lesions would enable one to better comprehend the chemical mechanistic pathways involved in their formation and in addition, these lesions can also serve as biomarkers of radiation-induced DNA damage.
As a result, assays have been developed to measure the yields of radiation-induced DNA damage with varying degrees of specificity, sensitivity and simplicity. To date, the principal assays used for the detection and quantification of specific DNA lesions have been the analytical techniques of gas chromatography or HPLC coupled with mass spectrometry or electro-chemical detection (3–5
) and other assays (7–9
). Depending on the lesions being assayed, each technique has its own advantages and disadvantages. The 32
P-post-labeling assay, as developed by Weinfeld et al.
), is a highly sensitive technique capable of detecting certain types of radiation-induced DNA damage products at the femtomole level and overcomes many of the problems previously encountered by the other techniques (6
The 32P-post-labeling assay involves the digestion of irradiated DNA by the action of three enzymes, namely, Snake venom phosphodiesterase (SVP), DNase I and shrimp alkaline phosphatase (SAP) as outlined in . This approach takes advantage of the fact that certain types of DNA lesions are refractory to these enzymes, preventing the cleavage of the internucleotide phosphodiester linkage immediately 5′ to the site of damage. This incomplete digestion of the irradiated DNA with these enzymes thereby yields dinucleoside monophophates containing these certain types of damage. These dinucleoside monophosphates are readily phosphorylated at their 5′-hydroxyl termini by polynucleotide kinase and [γ-32P]ATP. The unmodified bases are recovered as mononucleosides, and are not phosphorylated by the polynucleotide kinase. This technique has been used previously to measure lesions such as phosphoglycolates (pg) and thymine glycols (Tg) (), which are products of the indirect effect.
Strategy of post-labeling assay as modified from Weinfeld et al. and ‘X’ in the above represents all damaged nucleosides that are refractory to SVP digestion.
Chemical structures of lesions mentioned in this article.
The damage to cellular DNA arises from two sources, the direct effect and the indirect effect. Direct-type damage occurs when the ionizing energy is deposited in DNA itself or transferred into the DNA following ionization of the DNA solvation shell. The DNA solvation shell (Γ) consists of ~20–22 water molecules per nucleotide. Of these, ~15–17 water molecules associate with the DNA nucleoside while ~5 water molecules associate with the phosphate group (11
). These ~5 water molecules are tightly bound and difficult to remove. The water outside the solvation shell is termed as bulk water. The ionization of the DNA solvation shell produces a water radical cation and an electron. The water radical cation is then involved in two competing reactions: hole transfer to the DNA and the formation of OH•
via deprotonation. It has been shown that the formation of OH•
is not detected for Γ
9–10 but is detected for DNA with Γ
). The indirect-type damage occurs when the energy is deposited in the water surrounding the DNA (excluding the tightly bound water in the solvation shell) and the subsequent reaction of the radical products (OH•
), generated in the surrounding water, with DNA. As a point of emphasis, OH•
is not formed in DNA samples having Γ
Even though direct-type damage contributes to ~50% of the overall DNA damage (15
), it is not as well characterized, either quantitatively or mechanistically, as indirect-type damage. Consequently, a better understanding of the DNA lesions induced by the direct effect will lead to greater comprehension of the overall DNA damage chemistry as well as the relative biological effectiveness (RBE). The current study focuses on the direct-type lesions in DNA, comparing these with the indirect-type lesions of pg and Tg. The indirect-type damage product pg is formed when OH•
abstracts a hydrogen from C4′ of the DNA sugar moiety in the presence of oxygen (16
). The mechanism of Tg formation involves OH•
addition to the DNA base moiety in the presence of oxygen (17
). Thus, for both these radiogenic lesions, OH•
attack is required. Since the direct effect, at extremely low DNA hydration level of Γ
2.5, cannot involve the formation of OH•
, the formation of pg and Tg by this pathway is precluded. This assay, therefore, provides a means of comparing sugar and base damage products for the direct versus the indirect effect.
Three objectives were addressed in our present study: (i) detection of direct-type damage in the bases and deoxyribose groups and comparing these to indirect-type damage, (ii) determination of the influence of DNA hydration on the yields of direct-type damage and (iii) determination of the influence of irradiation temperatures on the yields of direct-type damage. These results provide new insights into the direct-type DNA damage chemistry.