UV-Induced Strand Breaks in Double-Stranded DNA Labeled with 5-Bromouracil: Frank or Secondary?

Abstract

Some literature reports suggest that in DNA labeled with 5-bromouracil (5BrU), near-UV photons lead to strand breaks that are formed due to the formation of a reactive uracil-5-yl radical capable of abstracting a hydrogen atom from its own or adjacent sugar moiety, which results in a direct strand break. However, other reports propose the formation of 2′-deoxyribonolactone rather than a strand break during the photodamage of 5BrU-substituted DNA. In order to resolve these contradictions, we carried out a series of experiments where 25 nucleotides-long DNA duplexes labeled with 5BrU were irradiated with 300 nm light. Two experimental methods were used to detect and separate the degradation products generated under experimental conditions, DHPLC (completely denaturing high-performance liquid chromatography) and denaturing PAGE electrophoresis. In addition, the identity of the particular products was confirmed with negative ion mass spectrometry. Our studies demonstrate that direct strand breaks reported in the past for 5BrU-labeled oligonucleotides are rather secondary breaks.