College of Chemistry, Fuzhou University, Fuzhou, 350108, China.
College of Chemistry, Fuzhou University, Fuzhou, 350108, China.
Biochem Biophys Res Commun. 2020 Apr 30;525(2):265-271. doi: 10.1016/j.bbrc.2020.02.062. Epub 2020 Feb 19.
The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5'-TTC-3'. PaRuvC uses Mg as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0-9.0. Elevated temperatures (37-60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system.
霍利迪交叉，一种四链 DNA 结构，是同源重组的重要中间产物。正确的霍利迪交叉解决是完成重组过程的关键。在大多数细菌细胞中，霍利迪交叉的切割主要由特定的内切酶 RuvC 完成。在这里，我们描述了一种机会性病原体铜绿假单胞菌（PaRuvC）中 RuvC 的生化特性和晶体结构。PaRuvC 特异性结合霍利迪交叉 DNA，并优先在 5'-TTC-3'处切割它。PaRuvC 使用 Mg 作为霍利迪交叉切割的首选二价金属辅因子，其最佳 pH 值为 8.0-9.0。较高的温度（37-60°C）可提高催化活性，但高于 53°C 的温度会降低蛋白质稳定性。我们在 2.4 Å 下测定了 PaRuvC 的晶体结构，并进行了突变分析，揭示了参与二聚体形成、底物结合和催化的关键残基。我们的研究结果有望为通过靶向其同源重组系统来对抗铜绿假单胞菌的抗生素耐药性提供有用信息。
