Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein–DNA complex remains active in the solid-state NMR rotor and that time-resolved ³¹P-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved ¹H-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.

中文翻译：

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通过实时 NMR 光谱监测 DNA 引物酶的初始引物合成

引物酶是 DNA 复制的关键酶，因为它们合成启动 DNA 复制所需的短引物。我们在此提出了溶液和固态的时间分辨核磁共振 (NMR) 光谱来研究古菌 pRN1 引物酶的初始二核苷酸形成反应。我们的研究结果表明，pRN1 引物酶的螺旋束结构域 (HBD) 准备了两种底物，然后将它们交给催化结构域以引发反应。通过使用三磷酸核苷酸类似物，反应速度大大减慢，使我们能够实时研究最初的二核苷酸形成。我们表明，沉积的蛋白质-DNA 复合物在固态 NMR 转子中保持活性，并且时间分辨³¹ P 检测的交叉极化实验可以监测二核苷酸形成的动力学。沉淀蛋白质样品的动力学与溶液态 NMR 测定的动力学相当。在快速魔角旋转频率 (100 kHz) 的时间分辨¹ H 检测实验中观察到引物合成过程中蛋白质构象的变化。 HBD 中聚集的大量光谱变化表明 HBD 对于定位核苷酸和二核苷酸的重要性。