Changes in DNA topology can regulate the dynamic equilibrium of r-loop, which is closely related to genome stability. DNA topoisomerase 1 (TOP1), as a highly conserved topoisomerase in eukaryotes, induces the accumulation of genomic R-loop (Camptothecin) with its specific inhibitor CPT (Camptothecin) and leads to genomic instability. However, it is still unknown how changes in genome topology affect the level of r-loop and thus regulate genome homeostasis.
On November 27, Qianwen Sun's lab in the School of Life Sciences at Tsinghua University published a paper titled "DNA polymerase harmonizes topological states and r-loops formation to" in the journal Nature Communications maintain genome integrity in arabidopsis**, revealing the molecular mechanism by which DNA polymerase in Arabidopsis thaliana is involved in regulating Topor-loop dynamics and DNA replication processes, thereby maintaining genome integrity.
The authors first used Top1 inhibitor (Top1I) inhibitor (TOP1I) in Arabidopsis thaliana to establish a monitoring system reflecting the level of R-loop and genome topology, and found that CPT treatment significantly promoted the increase of R-loop (R-loop change caused by topological structure change, referred to as Topor-loop) level and DNA break marker H2ax level in root tip tissue, and ultimately inhibited root growth. Reverse genetic screening by CPT showed that the mutation of DNA damage repair kinase ATM was particularly sensitive to CPT, and the accumulation of R-loop level in the genome was particularly significant, and the growth of roots was severely inhibited. Based on ATM mutants, this system was used to screen and identify mutations in the DNA polymerase catalytic subunit POL2A responsible for lead strand synthesis, which could restore the Topor-loop accumulation and DNA damage induced by TOP1I in ATM. Further studies have found that POL2A inhibits the accumulation of Topor-loops near the start site of DNA replication and reduces the level of DNA damage in cells undergoing DNA replication after CPT treatment. This study shows that DNA polymerases can synergistically regulate R-loop dynamics and DNA replication processes in response to changes in genome topology, thereby maintaining genome integrity. This finding provides important information for a deeper understanding of the mechanisms by which ATM deficiencies lead to resistance to top1I-targeted drugs during human cancer chemotherapy, and provides a possible new strategy for the combination of DNA-damaging drugs and stratification**.
Diagram of the working mode of ATM and DNA polymerase in synergistic regulation of R-loop dynamics and DNA replication process.
Qin Li, a 2016 Ph.D. from the School of Life Sciences, Tsinghua University, is the first author, and Associate Professor Sun Qianwen is the corresponding author. The research was supported by laboratory members Dr. Jincong Zhou, Dr. Shuai Li, Dr. Weifeng Zhang, Dr. Kuan Li, Yingxue Du, a 2017 PhD student, and Professor Yingxiang Wang from Fudan University. The research was supported by the National Natural Science of China, the Ministry of Science and Technology of the People's Republic of China, and the Tsinghua-Peking University Joint Center for Life Sciences.
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