Single-Molecule Analysis of Conformation Dynamics and Transition Kinetics of DNA Holliday Junction by Single-Molecule...

The relationship between conformational dynamics and multistate transition kinetics has been the fundamental focus for understanding the DNA Holliday junction (HJ) intermediated homologous genetic recombination process. Although the crystal structure and Markovian dynamics of HJ are known, the nature of the dynamics related to the transition frequency to thermodynamically stable multistate and intermediate states is not yet fully understood. Using single-molecule fluorescence resonance energy transfer (smFRET) and correlation based statistical analysis, we have identified intermittent conformational dynamics with three different patterns: periodic oscillation, correlated stochastic fluctuation, and damped oscillation. Furthermore, we found that the intermittently correlated stochastic fluctuation conformational dynamics of HJ goes through multistep transition kinetics along the transition coordinates, presenting an extreme pattern of conformational dynamics. There is also heterogeneity in the correlated stochastic fluctuation frequency due to heterogeneity in the molecular dynamics. Our results on conformational dynamics of HJ also support the multistate transition model. We applied multipoint time-correlation function analysis to smFRET efficiency time-trajectories resulting from Cy3/Cy5 labeled HJ to determine and compare the kinetics of various possible multistate conformational pathways. Results from the time correlation function (TCF) analysis show that intermediate conformational states between the energy states of two extreme conformations likely play a critical role in loop-gated conformational change mechanisms in HJ-mediated genetic recombination processes.