γ-Radiolysis and Hydroxyl Radical Produce Interstrand Cross-Links in DNA Involving Thymidine

Interstrand cross-links are minor components of the collection of products formed in DNA by ionizing radiation. Through their formation by other damaging agents, it is known that interstrand cross-links exert significant effects on replication and transcription. The structures of DNA interstrand cross-links produced as a result of gamma-radiolysis are unknown. Using synthetic duplexes we found that interstrand cross-link formation required thymidine and occurred with G values of approximately 10(-4) nmol J(-1). Enzymatic digestion of a tritiated substrate indicated that interstrand cross-links were derived from the reaction of 5-(2'-deoxyuridinyl)methyl radical (1) with the opposing 2'-deoxyadenosine to yield 5, which was identical to the product previously characterized when 1 was independently generated from a synthetic precursor. Conservative estimates indicated that 5 accounted for at least one-fourth of the interstrand cross-links produced in DNA by gamma-radiolysis. Utilization of a probe designed specifically to detect hole migration suggested that approximately 20% of the interstrand cross-links were produced by gamma-radiolysis via this pathway. Experiments using an independent source of hydroxyl radical indicated that cross-links were also produced by this species. Hence, DNA interstrand cross-links arising from 1 should result from a variety of oxidative stress mechanisms.