Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Nucleic Acids Res. 2022 Nov 28;50(21):12266-12273. doi: 10.1093/nar/gkac1101.
DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.
DNA 聚合酶 α（Polα）对于 DNA 复制的起始至关重要，并且对基因组诱变有显著贡献。然而，Polα 在 DNA 复制早期步骤中的活性和保真度尚未得到很好的研究。在这里，我们发现，在 DNA 合成的开始阶段，当延伸引物酶收到的 RNA 引物时，Polα 的突变率高于后期的 DNA 延伸步骤。动力学和结合研究表明，当 Polα 与嵌合 RNA-DNA 引物的核糖核苷酸相互作用时，其模板：引物的活性和亲和力显著更高。Polα 在 DNA 合成的前六个步骤中的活性变化很大，并且正确和错误 dNTP 掺入率的偏差导致保真度受损，尤其是在 RNA 引物延伸的第二步。此外，含有 RNA-DNA 引物的 Polα/模板：引物复合物的活性和稳定性增加，导致错配延伸的效率更高。
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