Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recognition by Damage Recognition Protein Rad4

How DNA-binding proteins search for and identify their target sites buried amid a vast excess of non-target sites remains a central question in biology. This study is aimed at elucidating the mechanism by which DNA damage recognition protein XPC recognizes diverse DNA lesions, and initiates DNA repair. Structural studies of Rad4 (yeast ortholog of XPC) bound to model lesions showed that DNA is unwound and bent at the lesion site and the damaged nucleotides are flipped out. Previous studies using laser T-jump perturbation approaches indicated that Rad4 interrogates potential sites by unwinding DNA on ~100-500 μs and eventually flips out damaged nucleotides on ~5-10 ms. However, these ‘ensemble’ measurements could not capture intrinsic DNA deformability that is potentially critical in the ability of Rad4/XPC to distinguish damaged from undamaged sites. Previous studies were also limited to measurements on short, linear DNA oligomers, while DNA in our cells is typically bent and supercoiled, which is expected to have a profound impact on DNA damage recognition. In this study, I employed an innovative fluorescence lifetime approach with fluorescent probes exquisitely sensitive to DNA twisting/bending fluctuations and mapped the range of conformations accessible to mismatched DNA substrates of varying Rad4-binding specificities. My results reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity mismatched DNA, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound. Conversely, nonspecific mismatched DNA retained a largely homogeneous, B-DNA-like conformation. Surprisingly, even mismatched-DNA specifically bound to Rad4 remained highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. Using T-jump, I unveiled the rates of intrinsic DNA conformational dynamics in mismatched DNA. Remarkably, these rates remained unaltered in the presence of Rad4. Finally, I examined the effect of DNA bending strain on the intrinsic deformability at the mismatched sites by extending the fluorescence lifetime studies in the context of DNA minicircles. These studies revealed amplified distortions for both matched and mismatched sites in DNA minicircles, with further distortions detected upon Rad4 binding to mismatched sites. Taken together, my results strongly suggest that intrinsic DNA deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites.