A new interdisciplinary study, recently published in Nucleic Acids Research (2025), uncovers how accessory proteins influence the growth of protein filaments essential for DNA repair. The research, led by Prof. Hung-Wen Li (Department of Chemistry) and Prof. Peter Chi (Institute of Biochemical Sciences), combines expertise in biophysics and biochemistry to investigate how RAD51, an enzyme central to homologous recombination, assembles on DNA during repair. Their collaboration reflects a growing trend of bridging the physical and life sciences to understand complex cellular mechanisms.
A key challenge in studying filament-forming proteins like RAD51 is identifying the functional unit by which these proteins extend on DNA, critical for understanding their assembly process. While structural techniques like cryo-electron microscopy and mass photometry have provided static snapshots or average sizes of RAD51 clusters, they cannot distinguish which oligomeric species are actively contributing during filament growth. This study directly tackled this problem by observing the real-time assembly of RAD51 on DNA under controlled force, allowing the authors to pinpoint the dynamic and functional extending units that drive recombination.
How can you watch protein filaments grow, one step at a time? The answer lies in a technique called optical tweezers, which uses highly focused laser beams to trap and manipulate tiny objects, like micron-sized beads attached to individual DNA molecules, without physically touching them. In this study, an individual DNA molecule was tethered between a surface and a micron-sized bead held in place by a laser. As RAD51 proteins assembled along the DNA, they stretched it, causing detectable changes in its end-to-end length. Optical tweezers can track these changes with nanometer precision, about one hundred-thousandth the width of a human hair, allowing researchers to “see” each step of protein binding in real time. This exquisite resolution makes optical tweezers uniquely suited for studying the dynamic mechanics of protein-DNA interactions at the single-molecule level.
Using this approach, the team monitored the stepwise growth of RAD51 filaments in real time, one assembly event at a time. Such precision revealed that RAD51, in the absence of regulatory factors, predominantly assembles in octameric units. However, when the accessory complex SWI5–SFR1 was introduced, the dominant step size shifted to tetramers, suggesting that SWI5–SFR1 remodels the oligomeric states of RAD51, reduces its dissociation, and promotes more stable and uniform filament assembly.
These findings shed light on how accessory proteins regulate the filament assembly process to ensure efficient DNA repair, an essential mechanism for maintaining genome integrity. By resolving the dynamic behavior of RAD51 at the molecular level, the study offers new insights into how recombination is regulated in cells, with implications for cancer biology and genome editing technologies. This work underscores the value of interdisciplinary research, specifically on the novel single-molecule platforms and in vitro biochemical works, in addressing key and fundamental biological questions. This work is supported by the National Science and Technology Council and National Taiwan University. The authors acknowledge the long-term support that allows the development of single-molecule microscopy tools and the success of this extensive collaborative effort.
SWI5–SFR1 reduces RAD51 recombinase extending units during filament assembly Open Access, Nucleic Acids Research, 53, gkaf676 (2025) https://doi.org/10.1093/nar/gkaf676
By Yingying Hu , Yen-Chan Chang , You-Yang Tsai , Hao-Yen Chang , Peter Chi , Hung-Wen Li
Prof. Petr Chi’s lab https://chi-lab.webflow.io/
Prof. Hung-Wen Li’s lab https://www.ch.ntu.edu.tw/hwli.html
(Top) High-resolution optical tweezers platform measures the functional extending units of RAD51 on DNA. (Bottom) Molecular model of accessory protein SS regulates RAD51 extending units and associated efforts in recombination.