为了促进DNA损伤耐受性的真核激活途径,PrimPol在聚合酶停滞障碍的下游合成从头寡核苷酸引物。这些引物使复制聚合酶能够恢复合成以确保及时完成DNA复制。从头开始合成需要单链DNA的协调,起始核苷酸,和PrimPol活性位点内的金属离子催化第一个磷酸二酯键的形成。在这里,我们研究人类PrimPol的催化结构域之间的相互作用,核苷酸,和DNA模板在每个催化步骤中确定引物合成的“编排”,其中底物以有序的方式结合。我们的发现表明,PrimPol进行从头引物合成的能力是由酶之间稳定相互作用的网络支撑的。模板,和核苷酸,正如我们之前观察到的相关启动酶CRISPR相关的Prim-Pol(CAPP)。一起,这些发现为人类PrimPol启动DNA合成建立了详细的模型,看起来非常保守。
To facilitate the eukaryotic repriming pathway of DNA damage tolerance,
PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within
PrimPol\'s active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human
PrimPol\'s catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the \'choreography\' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of
PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human
PrimPol, which appears highly conserved.