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“Flexible hinge” dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy

Abstract

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on similar timescales as one-dimensional (1D) diffusion of proteins on DNA if effective in stalling these proteins as they scan DNA. We examined the flexibility and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC). A previous Forster resonance energy transfer (FRET) study mapped DNA conformational distributions with cytosine analog FRET pair primarily sensitive to DNA twisting/unwinding deformations (Chakraborty et al. Nucleic Acids Res. 46: 1240–1255 (2018)). These studies revealed B-DNA conformations for nonspecific (matched) constructs but significant unwinding for mismatched constructs specifically recognized by Rad4, even in the absence of Rad4. The timescales of these unwinding fluctuations, however, remained elusive. Here, we labeled DNA with Atto550/Atto647N FRET dyes suitable for fluorescence correlation spectroscopy (FCS). With these probes, we detected higher FRET in specific, mismatched DNA compared with matched DNA, reaffirming unwinding/bending deformations in mismatched DNA. FCS unveiled the dynamics of these spontaneous deformations at ~ 300 µs with no fluctuations detected for matched DNA within the ~ 600 ns–10 ms FCS time window. These studies are the first to visualize anomalous unwinding/bending fluctuations in mismatched DNA on timescales that overlap with the < 500 µs “stepping” times of repair proteins on DNA. Such “flexible hinge” dynamics at lesion sites could arrest a diffusing protein to facilitate damage interrogation and recognition.

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Acknowledgements

We are deeply grateful to Marcia Levitus for her invaluable input throughout the design, development, and calibration of our home-built FCS apparatus. We also acknowledge a tremendous debt to Hans Frauenfelder, whose pioneering work on the existence of protein conformational substates opened the field of structure-dynamics-function of biomolecules.

Funding

This work was supported by grants from the American Heart Association (AHA-0730254 N) to S.V.K. and from the National Science Foundation (MCB-1715649 and MCB-2107527) to A.A.

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Correspondence to Anjum Ansari.

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Ten, T.B., Zvoda, V., Sarangi, M.K. et al. “Flexible hinge” dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy. J Biol Phys (2022). https://doi.org/10.1007/s10867-022-09607-x

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  • DOI: https://doi.org/10.1007/s10867-022-09607-x

Keywords

  • DNA unwinding/bending dynamics
  • Spontaneous fluctuations
  • Mismatched DNA dynamics
  • DNA damage recognition
  • Conformational energy landscape