Measurements of DNA Dynamics and Flexibility and Their Impact on Protein-DNA Interactions

DNA-protein interactions where DNA gets kinked, bent or otherwise deformed are vital for many biological processes. The flexibility and dynamics in these complexes remain poorly characterized because of the lack of adequate time resolution of many approaches. In this study, we use µs-resolved laser temperature-jump (T-jump) and fluorescence lifetime spectroscopies to measure DNA and protein-DNA conformational dynamics with high temporal resolution and sensitivity. We reveal novel findings on DNA dynamics in two main contexts: (1) bound to architectural proteins that facilitate chromatin packaging; and (2) when the DNA contains mismatches that are recognized by mismatch repair proteins. Architectural proteins from the HMGB family induce transient DNA kinks and enhance DNA flexibility to facilitate DNA packaging. How these proteins accomplish this DNA “softening” is not well understood. For example, it was not known whether the proteins bind to unbent DNA and then deform it, or if bent DNA conformations are “captured” by protein binding. Using an array of thermodynamic probes, we unveiled for the first time unbent DNA while bound to yNhp6A (yeast ortholog of HMGB), thus supporting the “bind-then-bend” mechanism. With T-jump, we measured DNA bending/unbending kinetics in the yNhp6A-DNA complex on timescales of ~0.5-1 ms, providing the first observation of DNA bending dynamics in complex with a nonspecific DNA-binding protein. DNA repair protein MutS is tasked with locating single mismatches (errors) introduced during replication and to initiate repair. These proteins must rapidly scan billions of base pairs of genomic DNA yet slow down to recognize mismatches—a daunting and puzzling task. What role DNA plays in facilitating mismatch recognition remains underexplored. We examined the conformations and dynamics of a series of mismatches repaired with different efficiencies. Weakly recognized substrates, e.g., T.T and T.C (in our sequence-context) were indistinguishable from the matched counterpart in their conformations/dynamics. In contrast, efficiently recognized mismatches induced severe disruptions in base stacking, exhibiting rapid (<10 µs) kinking fluctuations (e.g., +T) and/or shape distortions (e.g., G.T). These distortions/dynamics likely help stall a diffusing protein to facilitate recognition. How mismatch-induced DNA conformational distortions/dynamics, further modulated by sequence context, correlate with mismatch repair efficiencies remains to be explored.