Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), c/Nicolás Cabrera 1, Cantoblanco, 28049 Madrid, Spain.
Genes (Basel). 2021 Sep 24;12(10):1487. doi: 10.3390/genes12101487.
PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting synthesis of DNA primers, and especially when selecting the 3'nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3'-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs ddNTPs at the 3'elongation site, thus avoiding synthesis of abortive primers.
PrimPol 是在细胞核和线粒体中重新引发 DNA 复制所必需的，从而在复制应激期间促进叉的进展。ddC 是一种链终止核苷酸，已被广泛用于阻断线粒体 DNA 复制，因为它可以被复制聚合酶 Polγ 有效地掺入。在这里，我们表明，人类 PrimPol 在延伸引物穿过模板中的 8oxoG 损伤时会区分去氧核苷酸（ddNTP），但在开始 DNA 引物合成时也会区分，特别是在选择初始二聚体的 3'核苷酸时。与 dNTP 相比，即使在激活的锰离子存在下，PrimPol 掺入 ddNTP 的效率也非常低，只有 40 倍的 ddNTP 过量才会显著干扰 PrimPol 引发酶活性。这种对 ddNTP 的区分可防止引物过早终止，从而保证其用于延伸。人类 PrimPol 的晶体结构突出了 Arg 残基负责强的 dNTP/ddNTP 选择性，因为它与进入的脱氧核苷酸的 3'-OH 基团相互作用，而 ddNTP 中不存在该基团。Arg 在这里被证明对人类 PrimPol 的引发酶和聚合酶活性都至关重要，它有助于 dNTP 和 ddNTP 在 3'延伸位点的优先结合，从而避免合成无效的引物。
