Damaged DNA, generated by the abstraction of one of five hydrogen atoms from the 2′-deoxyribose ring of the nucleic acid, can contain a variety of lesions, some of which compromise physiological processes. Recently, DNA damage, resulting from the formation of a C3′-thymidinyl radical in DNA oligomers, was found to be dependent on nucleic acid structure. Architectures relevant to DNA replication were observed to generate larger amounts of strand-break and 1-(2′-deoxy-β-D-threo-pentofuranosyl)thymidine formation than that observed for duplex DNA. To understand how this damage can affect the integrity of DNA, the impact of C3′-thymidinyl radical derived lesions on DNA stability and structure was characterized using biophysical methods. DNA architectures evaluated include duplex DNA (dsDNA), single 3′ or 5′-overhangs (OvHgs), and forks. Thermal melting analysis and differential scanning calorimetry measurements indicate that an individual 3′-OvHg is more destabilizing than a 5′-OvHg. The presence of a terminal 3′ or 5′ phosphate decreases the to the same extent, while the effect of the phosphate at the ss-dsDNA junction of OvHgs is dependent on sequence. Additionally, the effect of 1-(2′-deoxy-β-D-threo-pentofuranosyl)thymidine is found to depend on DNA architecture and proximity to the 3′ end of the damaged strand. 1. Introduction Maintaining DNA integrity is essential to facilitate proper physiological processes including DNA replication, repair, and transcription. The disruption of replication by reactive oxygen species (ROS) has been linked to a variety of diseases and disorders [1–3]. Causative in the development of disease via this mechanism is the stalling or collapse of replication forks. When this occurs, atypical secondary structures form which must be resolved and/or repaired before replication can resume [4]. Additionally, it has been shown that elevated levels of oxidative stress during replication result in a prolonged S-phase and if left unrepaired, can lead to apoptosis [5]. Double strand-breaks (DSBs), being one of the most lethal DNA damaging events resulting from ROS, are known to form through oxidative damage and have recently been reported to be replication induced [6]. Replication induced DSBs are proposed to result from a single strand-break (SSB) and/or other damage lesions generated in replicating DNA [6]. Under conditions of oxidative stress, SSBs can result from hydrogen atom abstraction at the 2′-deoxyribose ring in oligonucleotides. When a single hydrogen atom is removed by a ROS from any of the five carbons of the sugar moiety
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