State Key Laboratory of Microbial Technology, Microbiology and Biotechnology Institute, Shandong University, 72 Binhai Road, Jimo, Qingdao, Shandong, 266237, P.R. China.
Department of Biochemistry, University of California, Riverside, CA 92521, USA.
Nucleic Acids Res. 2018 Jul 27;46(13):6627-6641. doi: 10.1093/nar/gky451.
PINA is a novel ATPase and DNA helicase highly conserved in Archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring in crystal and solution. The protein is able to promote Holliday junction (HJ) migration and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has direct physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues impaired the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA or ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest that SisPINA, Hjm and Hjc work together to function in replication fork regression, HJ formation and HJ cleavage.
PINA 是一种新型的 ATP 酶和 DNA 解旋酶，在古菌中高度保守，是生命的第三领域。来自 Sulfolobus islandicus 的 PINA（SisPINA）在晶体和溶液中形成六聚体环。该蛋白能够促进 Holliday 连接点（HJ）迁移，并与 HJ 特异性内切酶 Hjc 在物理和功能上相互作用。在这里，我们表明 SisPINA 与 Hjm（Hel308a）具有直接的物理相互作用，Hjm 是一种假定靶向复制叉的解旋酶。体外生化分析表明，Hjm、Hjc 和 SisPINA 能够协同协调 HJ 迁移和切割。缺失羧基 13 个氨基酸残基会损害 SisPINA 与 Hjm 之间的相互作用。晶体结构分析表明，羧基 70 个氨基酸残基折叠成 II 型 KH 结构域，在其他蛋白质中，该结构域的功能是结合 RNA 或 ssDNA。KH 结构域不仅介导 PINA 与 Hjm 和 Hjc 的相互作用，还调节 PINA 的六聚体组装。我们的研究结果表明，SisPINA、Hjm 和 Hjc 共同作用于复制叉后退、HJ 形成和 HJ 切割。
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