A cross-disciplinary team led by Professor Hung-Wen Li of the NTU Department of Chemistry and Professor Hung-Yuan Chi of the Institute of Biochemical Sciences has recently published its findings in the prestigious journal Nucleic Acids Research. The study provides an in-depth analysis of a critical step in DNA double-strand break repair—the mechanism by which the RAD51 protein assembles into filaments on DNA—and reveals how accessory proteins precisely regulate this process. By integrating biophysical and biochemical approaches, the research highlights the emerging trend of combining physical chemistry and life sciences to explore complex cellular mechanisms.

One of the major challenges in studying the dynamic process of protein assembly on DNA is identifying the “functional units” responsible for filament extension. Traditional methods such as cryo-electron microscopy can provide static structural information about RAD51 aggregates but cannot capture the functional minimal units during active filament assembly. This study overcame that limitation by employing a single-molecule mechanics platform, successfully observing and quantifying the stepwise assembly of RAD51 on DNA in real time and precisely defining its functional assembly units and regulatory mechanisms.

To achieve this, the team established an optical tweezers system with nanometer resolution. This technique uses a tightly focused laser beam to trap and apply force on micron-sized beads tethered to DNA molecules, thereby controlling DNA tension. In this setup, one end of the DNA molecule is held under constant tension. As RAD51 progressively binds to the DNA and forms filaments, the overall DNA length increases. A feedback control system enables the detection of nanometer-scale length changes—equivalent to one ten-thousandth the width of a human hair—thus allowing researchers to “see” each binding event.

Using this platform, the researchers quantified the stepwise assembly process of RAD51 filaments and observed how it is regulated by accessory proteins. The experiments revealed that in the absence of accessory proteins, RAD51 extends primarily in octamer units. However, when the SWI5-SFR1 accessory protein was added, the extension units shifted to tetramers. This demonstrates that SWI5-SFR1 alters the oligomeric state of RAD51 in solution and significantly reduces its probability of dissociating from DNA, thereby promoting more stable and consistent filament assembly.

The study provides direct evidence of how accessory proteins regulate the kinetics of RAD51 assembly, ensuring both the efficiency and accuracy of DNA repair—a process essential for maintaining genome stability. By capturing RAD51 dynamics at the single-molecule level, the work offers fresh insights into the molecular mechanisms of homologous recombination and DNA repair, with potential implications for fields such as cancer biology and genome editing.

This achievement underscores the importance of cross-disciplinary collaboration, combining state-of-the-art single-molecule biophysical platforms with advanced biochemical methods to open new frontiers in basic life science research. The team also expressed gratitude for the long-term support of the National Science and Technology Council (NSTC) and NTU, which enabled the establishment of single-molecule fluorescence and force spectroscopy platforms that were critical to the success of this study.