NTU Team Unraveled the Repair Mechanism for DNA Replication Published in Nature Communications

DNA replication is crucial for the development and the growth of organisms, and for organs to maintain a constant number of cells. Errors occurring during DNA replication will lead to genetic mutations, which may evolve into cytometaplasia, or even cancer, hence cells have developed a set of protective mechanisms. When DNA is damaged, or encounters obstacles that induce replication stress, the replication fork will enter a stalled state, and regress into a special DNA structure (reversed fork), in order to protect itself from breaking. At the same time, the RAD51 recombinase accumulates at the site of the damage, and forms a nucleoprotein filament polymer on the single-stranded DNA (ssDNA) of the reversed fork, in order to protect the newly synthesized DNA from nuclease degradation, and to restart DNA replication. However, in the entire course of reaction, because of the abundance of RPA protein complex within cells, and because of its high binding affinity to ssDNA, it will hinder the binding of RAD51 to single-stranded DNA. The mechanism regulating the formation of RAD51 nucleoprotein filaments on reversed fork still has many unknowns.

In previous studies, the research team found that the CST (CTC1-STN1-TEN1) protein complex played the role of protecting the reversed fork and regulating RAD51. In this study, the team with diverse expertise used complementary technologies to clarify the detailed cellular mechanism in regulating replication stress, from the cellular level, to the analysis of biochemical functions of proteins and DNA, and to observations and measurements at the single-molecule level. Cellular imaging experiments showed that under replication stress, both RPA and CST protein would gather on the stalled replication fork. The first author of this paper, Dr. Kai-Hang Lei (李啟恆), though the use of different protein expression systems and protein purification strategies, succeeded in obtaining highly purified CST, RPA, and RAD51 recombinant proteins for further analysis of the biochemical properties in their interactions. Results showed that CST and RPA proteins could co-occupy the same ssDNA, and CST interacted with RAD51 directly at the protein to protein level, bringing RAD51 to RPA-bound ssDNA. Meanwhile, Han-Lin Yang, a master degree student and the co-author of the paper, used single-molecule fluorescence experiments to further prove that CST and RPA indeed coexisted on the same ssDNA. This study clarified that RPA and CST, two proteins with multiple OB-fold domains but with different DNA binding affinities, competed with each other, so that both proteins could co-occupy the same DNA molecule (Figure 1). Since an abundance of RPA in cells inhibited RAD51 from binding to the reversed fork, the ability of CST and RPA to coexist would assist RAD51 to accumulate on DNA and to form nucleoprotein filaments, thereby promoting subsequent DNA replication repair (Figure 2).

CST mutations lead to growth retardation, bone marrow failure, neurological disorder, liver fibrosis, cancer and other diseases. This study found that CST played an important role in DNA replication and elucidated possible causes for these diseases, and offered further information for future diagnosis and treatment. In addition, cancer is characterized by rapid growth that accumulates much DNA replication stress. If in certain types of cancer, CST mutations and the inability to repair DNA under replication stress normally are detected, effective targeted therapy may be developed to treat the causes that result in replication pressure.

The full text of the paper is published in Nature Communications. The results of this study once again demonstrate the importance of interdisciplinary teamwork. Complementary research expertise accelerates research progress, and generates more in-depth discussions on the issues involved through different perspectives. This study was conducted with the support of the Ministry of Science and Technology, the National Taiwan University, and the Academia Sinica. The team led by Professor Hung-Yuan (Peter) Chi (冀宏源), the Institute of Biochemical Sciences, the National Taiwan University (Kai-Hang Lei李啟恆, Hao-Yen Chang張皓衍, and Hsin-Yi Yeh葉欣怡), the team led by Professor Hung-Wen Li (李弘文), the Department of Chemistry, the National Taiwan University (Han-Lin Yang楊翰霖 , Tzu-Yu Lee李子于), and the team led by Professor Weihang Chai in the United States (Dinh Duc Nguyen, Xinxing Lyu, Megan Chastain), have jointly authored this paper.

Full text of the paper: https://www.nature.com/articles/s41467-021-26624-x

Web page for Professor Hung-Yuan (Peter) Chi, the Institute of Biochemical Sciences, the National Taiwan University: http://ibs.ntu.edu.tw/about/staffDetail/49

Web page for Professor Hung-Wen Li, the Department of Chemistry, the National Taiwan University: https://www.ch.ntu.edu.tw/member/faculty/hwli/

Web page of Professor Weihang Chai : https://weihangchai.wixsite.com/chailab