Negative supercoiling facilitates human TOP3α-mediated decatenation of DNA braids via catenation junction melting
- 1DNA Motors Group, MRC Laboratory of Medical Sciences, London W12 0HS, UK
Abstract
DNA catenation between sister chromatids arises during replication and must be efficiently resolved to ensure faithful chromosome segregation. The removal of DNA catenation is generally attributed to the type II topoisomerase TOP2, which catalyses double-stranded DNA passage reactions and can therefore directly remove inter-DNA linkages. By contrast, the type IA topoisomerase TOP3 catalyses strand passage through transient breaks in single-stranded DNA. Here, using single-molecule optical tweezers and fluorescence microscopy, we show that negative supercoiling promotes local melting of duplex DNA at DNA-DNA crossings within braided molecules, generating single-stranded DNA that can be engaged by TOP3. These supercoiled substrates can be efficiently resolved by TOP3-RMI1-RMI2 (TRR), whereas relaxed DNA is resistant to TRR decatenation. Moreover, we find that RMI2 and the ssDNA binding Replication Protein A (RPA) stimulates TOP3 decatenation and demonstrate that decatenation remains viable up to 30pN of tension on the substrate. These findings reveal a mechanism by which torsional stress converts an otherwise inaccessible duplex DNA crossing into a substrate for a single-strand-specific topoisomerase. We propose that negative supercoiling can therefore direct type IA topoisomerases to topological DNA linkages by promoting local duplex melting, providing a physical mechanism that couples DNA supercoiling to the resolution of DNA entanglements.
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BibTeX
@misc{saravanan2026negative,
title = {{Negative supercoiling facilitates human TOP3α-mediated decatenation of DNA braids via catenation junction melting}},
author = {S. Saravanan and A. I. Koetje and G. A. King and D. S. Rueda and L. Aragon},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.23.753725},
url = {https://www.biorxiv.org/content/10.64898/2026.09.23.753725v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Negative supercoiling facilitates human TOP3α-mediated decatenation of DNA braids via catenation junction melting
AU - S. Saravanan
AU - A. I. Koetje
AU - G. A. King
AU - D. S. Rueda
AU - L. Aragon
PY - 2026
DA - 2026/09/23
PB - bioRxiv
DO - 10.64898/2026.09.23.753725
UR - https://www.biorxiv.org/content/10.64898/2026.09.23.753725v1
AB - DNA catenation between sister chromatids arises during replication and must be efficiently resolved to ensure faithful chromosome segregation. The removal of DNA catenation is generally attributed to the type II topoisomerase TOP2, which catalyses double-stranded DNA passage reactions and can therefore directly remove inter-DNA linkages. By contrast, the type IA topoisomerase TOP3 catalyses strand passage through transient breaks in single-stranded DNA. Here, using single-molecule optical tweezers and fluorescence microscopy, we show that negative supercoiling promotes local melting of duplex DNA at DNA-DNA crossings within braided molecules, generating single-stranded DNA that can be engaged by TOP3. These supercoiled substrates can be efficiently resolved by TOP3-RMI1-RMI2 (TRR), whereas relaxed DNA is resistant to TRR decatenation. Moreover, we find that RMI2 and the ssDNA binding Replication Protein A (RPA) stimulates TOP3 decatenation and demonstrate that decatenation remains viable up to 30pN of tension on the substrate. These findings reveal a mechanism by which torsional stress converts an otherwise inaccessible duplex DNA crossing into a substrate for a single-strand-specific topoisomerase. We propose that negative supercoiling can therefore direct type IA topoisomerases to topological DNA linkages by promoting local duplex melting, providing a physical mechanism that couples DNA supercoiling to the resolution of DNA entanglements.
ER -