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Title: Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

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 ofmore » Rad4/XPC to recognize many structurally dissimilar lesions« less

Authors:
 [1];  [2];  [1];  [3];  [3];  [1];  [1]
  1. Univ. of Illinois, Chicago, IL (United States)
  2. National Inst. of Health (NIH), Bethesda, MD (United States)
  3. New York Univ. (NYU), NY (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)
Sponsoring Org.:
USDOE; National Science Foundation (NSF); National Institutes of Health (NIH)
OSTI Identifier:
1543988
Grant/Contract Number:  
MCB060037
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 46; Journal Issue: 3; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology

Citation Formats

Chakraborty, Sagnik, Steinbach, Peter J., Paul, Debamita, Mu, Hong, Broyde, Suse, Min, Jung-Hyun, and Ansari, Anjum. Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex. United States: N. p., 2017. Web. doi:10.1093/nar/gkx1216.
Chakraborty, Sagnik, Steinbach, Peter J., Paul, Debamita, Mu, Hong, Broyde, Suse, Min, Jung-Hyun, & Ansari, Anjum. Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex. United States. doi:10.1093/nar/gkx1216.
Chakraborty, Sagnik, Steinbach, Peter J., Paul, Debamita, Mu, Hong, Broyde, Suse, Min, Jung-Hyun, and Ansari, Anjum. Mon . "Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex". United States. doi:10.1093/nar/gkx1216. https://www.osti.gov/servlets/purl/1543988.
@article{osti_1543988,
title = {Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex},
author = {Chakraborty, Sagnik and Steinbach, Peter J. and Paul, Debamita and Mu, Hong and Broyde, Suse and Min, Jung-Hyun and Ansari, Anjum},
abstractNote = {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},
doi = {10.1093/nar/gkx1216},
journal = {Nucleic Acids Research},
number = 3,
volume = 46,
place = {United States},
year = {2017},
month = {12}
}

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