Single-molecule binding characterization of primosomal protein PriA involved in replication restart

文献情報

出版日 2021-05-25

DOI 10.1039/D1CP00638J

インパクトファクター 3.676

著者

Tzu-Yu Lee, Yi-Ching Li, Min-Guan Lin, Chwan-Deng Hsiao, Hung-Wen Li

原文を見る

要旨

DNA damage leads to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes the DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplexed leading strand at sub-nanomolar affinity (Kd = 268 ± 99 pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as a monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.

掲載誌

Physical Chemistry Chemical Physics

CiteScore: 5.5

自己引用率: 10.3%

年間論文数: 3036

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