Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.

Primase 是 DNA 复制机制的重要组成部分，负责合成 RNA 引物，从而启动前导链和滞后链 DNA 合成。细菌引发酶活性可以通过饥饿诱导核苷酸 (p)ppGpp 进行调节。该调节有助于及时抑制革兰氏阳性细菌枯草芽孢杆菌中氨基酸饥饿时的 DNA 复制。在这里，我们描述了 (p)ppGpp 对B. subtilis DnaG primase体外 活性的影响。使用单核苷酸分辨率引发酶测定，我们剖析了 ppGpp 对枯草芽孢杆菌起始、延伸和保真度的影响初级酶。我们发现 ppGpp 对起始有轻微影响，但强烈抑制引物延伸并降低引物持续合成能力，从而促进引物延伸的终止。高 (p)ppGpp 浓度与低 GTP 浓度一起抑制引物酶活性。这解释了在饥饿期间对复制延伸的强烈抑制，这会导致枯草芽孢杆菌中高水平的 (p)ppGpp 和 GTP 的消耗。最后，我们发现降低 GTP 浓度会导致引物碱基配对不匹配，从而允许引发通读，并且 ppGpp 会减少通读以保护引发保真度。这些结果突出了 (p)ppGpp 在环境压力开始时在波动的核苷酸浓度中保护复制体完整性和基因组稳定性方面的重要性。