The Effect of Tension on Closely Spaced Nicks in Naked DNA Molecules

Cells always protect their genomic integrity from environmental stresses and harmful chemicals produced during metabolism. Double-stranded breaks(DSB) in DNA caused by factors such as free radicals or ionizing radiation play an important role in triggering DNA repair pathways and apoptosis. These DSBs are often formed by closely spaced nicks. Although methods exist to quantify single nicks on plasmids, the detection of multiple nicks or clustered nicks on DNA molecules is not so straight forward. By stretching single DNA molecules in low ionic strength buffers, sections containing clustered nicks denature locally and induce DSB. The detection of closely spaced nicks on double-stranded (ds) DNA is done by applying tension on DNA molecules with dual-beam optical tweezers and notating the time at which the DNA breaks in relation to the starting time (sustaining time). The effects of nicks caused by the sample preparation process and oxidative agents in the buffer were investigated. DNA sustaining time under low force (4∼6 pN) is around 50 minutes in low salt buffer. Higher forces (14∼16 pN) cause the sustaining time to shift toward a shorter time frame (∼30 minutes). The sustaining time of DNA molecules prepulled to a predetermined maximum force (14∼16, 24∼26, or 34∼36 pN) also show shifts toward shorter time.