Mayle Ryan, Georgescu Roxana, O'Donnell Michael E
HHMI and The Rockefeller University, New York, NY 10065.
Proc Natl Acad Sci U S A. 2025 Sep 16;122(37):e2517556122. doi: 10.1073/pnas.2517556122. Epub 2025 Sep 10.
Replication of cellular chromosomes requires a primase to generate short RNA primers to initiate genomic replication. While bacterial and archaeal primase generate short RNA primers, the eukaryotic primase, Polα-primase, contains both RNA primase and DNA polymerase (Pol) subunits that function together to form a >20 base hybrid RNA-DNA primer. Interestingly, the DNA Pol1 subunit of Polα lacks a 3'-5' proofreading exonuclease, contrary to the high-fidelity normally associated with DNA replication. However, Polδ and Polε synthesize the majority of the eukaryotic genome, and both contain 3'-5' exonuclease activity for high fidelity. Nonetheless, even the small amount of DNA produced by Pol1 in each of the many RNA/DNA primers during chromosome replication adds up to tens of millions of nucleotides in a human genome. Thus, it has been a longstanding question why Pol1 lacks a proofreading exonuclease. We show here that Polα is uniquely capable of traversing common oxidized or hydrolyzed template nucleotides and propose that Polα evolved to bypass these common template lesions when they are encountered during chromosome replication. Additionally, we show a unique ability of replication factor C (RFC) to stimulate Polδ lesion bypass, independent of its sliding clamp. This suggests that there may be a coordination between Polδ and RFC that does not involve RFC loading of PCNA.
细胞染色体的复制需要一种引发酶来生成短RNA引物以启动基因组复制。虽然细菌和古细菌的引发酶会生成短RNA引物，但真核生物的引发酶——DNA聚合酶α-引发酶（Polα-primase），包含RNA引发酶和DNA聚合酶（Pol）亚基，它们共同作用形成一个大于20个碱基的杂交RNA-DNA引物。有趣的是，Polα的DNA聚合酶1（Pol1）亚基缺乏3'-5'校对核酸外切酶，这与通常与DNA复制相关的高保真度相反。然而，DNA聚合酶δ（Polδ）和DNA聚合酶ε（Polε）合成了真核生物基因组的大部分，并且两者都具有用于高保真度的3'-5'核酸外切酶活性。尽管如此，即使在染色体复制过程中，Pol1在众多RNA/DNA引物中的每一个中产生的少量DNA，在人类基因组中加起来也有数千万个核苷酸。因此，长期以来一直存在一个问题，即为什么Pol1缺乏校对核酸外切酶。我们在此表明，Polα具有独特的能力来穿越常见的氧化或水解模板核苷酸，并提出Polα在染色体复制过程中遇到这些常见模板损伤时进化出绕过它们的能力。此外，我们展示了复制因子C（RFC）刺激Polδ绕过损伤的独特能力，这与其滑动夹无关。这表明Polδ和RFC之间可能存在一种不涉及RFC加载增殖细胞核抗原（PCNA）的协调。
