TUE-CMS

Abstract: Single-molecule measurements can resolve the complexity that is inherent in complex energy landscapes but obscured in ensemble experiments because of sample averaging. The recent development of SM-Fluoresce techniques with force spectroscopy enable us to go beyond the ensemble average and measure the behavior of individual biomolecules. These approaches can directly resolve transient intermediate states and multiple reaction pathways thus are uniquely powerful in characterizing the complex dynamics biological processes. Here, using single-molecule FRET coupled optical tweezer technique; we reveal previously hidden conformations and the chaotic behavior during the branch migration of Holiday Junction.

The Holliday junction (HJ), a central intermediate in various genetic processes, where branch migration allows the exchange between homologous DNA regions. However, the detailed mechanism for this key step of DNA recombination is unidentified. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg 2+ concentration as well as the amount of external force applied. Here, we provide a quantitative method to analyse single-molecule fluorescence resonance energy transfer (smFRET) data, thereby probing the isomerization dynamics of Holliday junctions, which display such heterogeneous dynamics over a long observation time. We have monitored the steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. Mg 2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs. We have carried out a nonlinear time series analysis of the FRET time series data. The phase space plots very clearly reveal the emergence of two lobes as the Mg 2+ concentration is increased. The power spectrum obtained from the fast fourier transform also reveal a power law behavior indicating a self similar behavior.