Title: Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.
Authors: Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse; Min, Jung-Hyun; Ansari, Anjum
Published In Nucleic Acids Res, (2018 02 16)
Abstract: Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, 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; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating 'conformational capture' of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.
PubMed ID: 29267981
MeSH Terms: Binding Sites; DNA Damage; DNA Repair*; DNA, Fungal/chemistry*; DNA, Fungal/genetics; DNA, Fungal/metabolism; DNA-Binding Proteins/chemistry*; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Fluorescent Dyes/chemistry; Gene Expression; Kinetics; Molecular Dynamics Simulation; Nucleic Acid Conformation; Oligodeoxyribonucleotides/chemical synthesis; Oligodeoxyribonucleotides/metabolism; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Saccharomyces cerevisiae Proteins/chemistry*; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism; Saccharomyces cerevisiae/chemistry*; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae/metabolism; Spectrometry, Fluorescence/methods; Spectrometry, Fluorescence/statistics & numerical data; Substrate Specificity