RPA-ssDNA co-phase separation facilitates RAD51 enrichment during homologous recombination

  • Nucleic Acids Res. 2026 Jun 8;54(11):gkag586. doi: 10.1093/nar/gkag586.
Yanan Li  1  2 ,  Yi Zhao  2 ,  Yinghong Chen  3  4 ,  Teng Wang  2  5 ,  Lulu Bi  2  6 ,  Yanling Bao  2 ,  Lishuang Chen  2 ,  Xia Zhang  2 ,  Bingkai Cheng  2 ,  Meng Hu  2 ,  Shengli Jing  1 ,  Chao Liu  3 ,  Wei Li  3 ,  Bo Sun  2
Affiliations
  • 1. College of Life Sciences, Xinyang Normal University, Xinyang, Henan 464000, China.
  • 2. School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
  • 3. Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou,Guangdong 510623,China.
  • 4. Department of Basic Medical Sciences, Shantou University Medical College, Shantou, Guangdong 515041, China.
  • 5. Department of Medical Laboratory, Jining No. 1 People's Hospital, Jining, Shandong 272011, China.
  • 6. Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China.
PMID: 42273915 DOI: 10.1093/nar/gkag586
Abstract

Homologous recombination (HR) is pivotal for the maintenance of genome integrity. During HR, replacing replication protein A (RPA) with the recombinase RAD51 on single-stranded DNA (ssDNA) is crucial in forming the presynaptic complex for homology search. However, how RAD51 identifies legitimate RPA-coated ssDNA involved in HR to prepare for the replacement remains elusive. Here, we develop an innovative approach to generate ssDNA for the single-molecule measurements of this transaction. We find that human RPA can undergo phase separation and co-condense with coated ssDNA, a process primarily mediated by the N-terminal DNA-binding domain of RPA70 (RPA70N). The resulting RPA-ssDNA co-condensates form stable nucleoprotein assemblies that withstand disruptive forces of tens of piconewtons. Intriguingly, both in vitro and in vivo evidence indicates that these co-condensates act as hubs for the local enrichment of RAD51. Consequently, deletion or sequestration of RPA70N readily suppresses the phase separation ability of RPA, reduces RAD51 enrichment, and ultimately compromises DNA double-strand break (DSB) repair. Our work defines the key determinants underlying RPA-mediated recombinase enrichment during the early stage of HR-directed DSB repair.