Deformability and Dynamics in Linear and Looped/Supercoiled DNA with Implications for Damage Sensing

Damage-sensing proteins must scan billions of base pairs (bp) of genomic DNA to identify damaged sites and initiate repair. These proteins diffuse rapidly along DNA, with <500 µs “dwell” time per site, while damage recognition—often involving DNA bending or unwinding—occurs over much slower timescales (>10 ms). This discrepancy in “interrogation” and recognition timescales raises a key question: how do proteins slow down to recognize damage? Additionally, most studies use short, torsionally-relaxed DNA, but in cells, DNA is often looped and/or supercoiled, introducing bending and torsional strain. These topological changes are expected to impact DNA deformability and damage recognition. We employed advanced fluorescence approaches to study protein-DNA dynamics on biologically relevant timescales, identified DNA features enabling damage recognition, and unveiled how looping/supercoiling alters DNA mechanics and protein binding. We studied two repair proteins: 1) Rad4 (yeast ortholog of human XPC), which recognizes DNA lesions from UV-light and other environmental genotoxins and initiates nucleotide excision repair; 2) MutS, which recognizes single mismatches or unpaired nucleotides introduced during replication and initiates mismatch repair. Using linear DNA with 3-bp mismatches as mock lesions for Rad4, we found that Rad4-specific substrates were more dynamic on sub-millisecond timescales, similar to Rad4 dwell times, suggesting that these fluctuations help “stall” Rad4 to allow recognition. These substrates also showed slower (>50 ms) fluctuations, potentially reflecting spontaneous sampling of Rad4-preferred conformations, supporting a “conformational capture” mechanism. To study the effects of DNA topology, we used torsionally-relaxed and supercoiled DNA minicircles (126 bp and 336 bp, comparable in size to ~150 bp DNA persistence length). In 126-bp relaxed minicircles, Rad4 affinity increased ~50-fold and MutS affinity ~10-fold, to their respective specific sites. Interestingly, DNA appeared more rigid in these minicircles compared to linear DNA. 336-bp minicircles also impacted DNA conformations, but only when supercoiled. This study is the first to show how bending strain enhances damage recognition and how looping/supercoiling influences DNA conformations at mismatched sites. It emphasizes that short, linear DNA fragments are poor models for in vivo damage sensing and highlights the need for further studies exploring the role of DNA topology in repair mechanisms.

Author dc:creator

• Saroj Baral (23291287)

• Biophysics
• General

Statement dc:rights

• In Copyright

DOI dc:identifier

https://doi.org/10.25417/uic.31451029.v1

OAI identifier oai:identifier

oai:figshare.com:article/31451029