Single-molecule biophysics of DNA cyclization

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Jeong, Jiyoun
Kim, Harold D.
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Structure and dynamics of DNA impact how the genetic code is processed and maintained. Thus, understanding the physical properties of DNA is of fundamental importance to biology. The physical properties of DNA have been extensively studied by a DNA cyclization assay, which measures the probability of intramolecular end-closure of DNA mediated by cohesive single-stranded overhangs called sticky ends. DNA cyclization kinetics are thought to have a clear theoretical basis, however, previous experimental studies produced conflicting results especially at lengths around 100 bp where DNA requires strong bending. The conflict partially arises from the incomplete understanding of how the intramolecular sticky-end joining reaction is influenced by the energetics and geometry of cyclized DNA. The main aim of this thesis is to bridge the gap between experimental measurables and theoretical predictions of DNA cyclization by investigating the association and dissociation kinetics of sticky ends of various DNA substrates. Using single-molecule fluorescence resonance energy transfer (FRET), we first show how the cyclization probability of short DNA depends on the rotational positioning of sticky ends around the helical axis. We find that rotational positions of sticky ends do not affect the cyclization rate, but allow the decyclization rate to oscillate, which we show to be related to the stackability of sticky ends at their termini. We also explore the effect of mismatches on DNA cyclization. Here, we present counter-intuitive findings that, for DNA cyclized by stackable sticky ends, base pair mismatch inside the DNA loop decreases the loop lifetime despite reducing the overall bending stress. This unexpected effect is contingent upon the terminal stackability of sticky ends and is most prominent for a central mismatch, furthest from the joined sticky ends. These findings show that base pair mismatch transfers bending stress to the antipodal side of the loop through an allosteric mechanism known as cooperative kinking. Based on our findings, we present a three-state model that correctly explains the apparent kinetics of DNA cyclization and decyclization.
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