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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: DNA Repair (Amst). 2021 Jul 29;107:103194. doi: 10.1016/j.dnarep.2021.103194

Fig.7.

Fig.7.

Proposed DNA ‘opening’ trajectory and ‘kinetic gating’ mechanism of Rad4/XPC. The top panel illustrates distinct binding modes for Rad4/XPC as it searches for, interrogates, and recognizes a damaged site, and the time scales for fluctuations between these modes, based on prior studies [27, 29, 30]. The middle panel shows a schematic free-energy profile along the ‘opening’ trajectory. The faster 100- to 500-μs nonspecific untwisting step entails a smaller energetic barrier than the slower 5-to 10-ms rate-limiting step (‡) of the ‘opening’ process. The rate-limiting step involves sufficiently unwound and bent DNA but with the nucleotides not yet fully or stably flipped out into the BHD2/BHD3 groove [29]. The free energy barrier (ΔG‡ opening) for ‘opening’ damaged DNA (red) is naturally lower than that for undamaged DNA (green) as DNA damage usually destabilizes the B-DNA structure. For Rad4 mutants that are lacking either β-hairpin2 or β-hairpin3, the protein can still overcome ΔG‡ opening to form ‘open-like’ structures [27, 28]. The bottom panel illustrates that, for each step along the ‘opening’ trajectory, there is also a kinetically competing process of diffusion of Rad4/XPC along the DNA, characterized by ΔG‡ diffusion. For undamaged DNA, the high ΔG‡ opening compared with ΔG‡ diffusion favors the protein diffusing away before ‘opening’ a given site, while for damaged DNA this competition favors ‘opening’. When the diffusion of the protein is blocked (e.g., by tethering), DNA containing consecutive C/G’s (CCC/GGG) could be ‘opened’ indicating that the ΔG‡ opening is thermally surmountable and that the ‘open(-like)’ structure was thermodynamically the most stable structure for such DNA. However, this study showed that this is not the case for DNA containing alternating CG/GC repeats: for this DNA, the ‘open(-like)’ structure is no longer the most stable structure, and the DNA retains predominantly B-DNA like conformation even under tethered conditions. We note here that while the free energies of the transition state ensemble (‡) are shown to be approximately the same for all DNA constructs, we cannot rule out that this free energy gets successively larger as we go from damaged to ‘openable’ CCC/GGG to ‘open-resistant’ CGC/GCG.