Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.
Biochem J. 2020 Oct 16;477(19):3911-3922. doi: 10.1042/BCJ20200269.
DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein - a 3' to 5' superfamily-2 DNA helicase - is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that the presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.
DNA 复制叉经常会遇到模板 DNA 损伤，从而导致其复制进程停滞。PriA 依赖性途径是革兰氏阳性菌中主要的复制起始机制，它需要几种引发酶蛋白。其中，PriA 蛋白——一种 3' 到 5' 超家族 2 DNA 解旋酶——是识别 DNA 损伤的关键因素，它还能招募其他蛋白。在这里，我们通过生化和动力学分析研究了肺炎链球菌 PriA（SpPriA）的 ATP 酶和解旋酶活性。通过比较各种 DNA 底物，我们观察到 SpPriA 无法解开具有高 GC 含量的双链 DNA。我们构建了一个缺失突变蛋白（SpPriAdeloop），其中 PriA 的 DNA 结合域的环区已被去除。对 SpPriAdeloop 的功能分析表明，环区对于赋予解旋酶的 DNA 结合特性很重要。我们还表明，DnaD 加载蛋白的存在对于增强 SpPriA 的 ATP 酶和 DNA 解旋活性很重要。
