A cross-disciplinary team led by Professor Hung-Wen Li from the Department of Chemistry and Professor Hung-Yuan Chi from the Institute of Biochemical Sciences at National Taiwan University (NTU) recently published their 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 assembly of RAD51 protein into filaments on DNA—and uncovers how accessory proteins precisely regulate this process. By combining biophysical and biochemical approaches, the research exemplifies the modern trend of integrating physical chemistry with life sciences to investigate complex cellular mechanisms.

A major challenge in studying the dynamic assembly of proteins on DNA is identifying the “functional units” responsible for filament extension. While methods such as cryo-electron microscopy can provide static structural information on RAD51 aggregates, they cannot reveal the minimal functional units during DNA assembly. This study overcame that limitation by employing a single-molecule mechanics platform, allowing real-time observation and quantification of RAD51’s stepwise assembly on DNA and enabling precise definition of its functional assembly units and regulatory mechanisms.

To achieve this, the team developed a nanometer-resolution optical tweezers system. The technique uses a tightly focused laser beam to trap and apply force to micron-sized beads attached to DNA molecules, thereby controlling DNA tension. In this setup, one end of the DNA is held under constant tension. As RAD51 binds and forms filaments, the overall DNA length increases. A feedback control system detects nanometer-scale length changes—equivalent to one hundred-thousandth the width of a human hair—allowing researchers to “see” each protein binding event in real time.

Using this platform, the team quantified the stepwise assembly of RAD51 filaments and examined how accessory proteins influence this process. The experiments showed that without accessory proteins, RAD51 extends primarily in octamer units. When the SWI5-SFR1 accessory protein is present, the extension unit shifts to tetramers. This indicates that SWI5-SFR1 alters RAD51’s oligomeric state in solution and significantly reduces its probability of dissociation from DNA, promoting a more stable and uniform filament assembly.

The study provides concrete insights into how accessory proteins regulate RAD51 assembly kinetics, ensuring high efficiency and accuracy in DNA repair—a process critical for genome stability. Observing RAD51 behavior at the single-molecule level offers new perspectives on the molecular mechanisms of homologous recombination and DNA repair and has potential implications for cancer biology and genome editing.

This work highlights the value of cross-disciplinary collaboration, combining state-of-the-art single-molecule biophysical platforms with advanced biochemical techniques to advance fundamental life sciences 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 microscopy platforms critical to this research.