DNA Replication Laboratory, Howard Hughes Medical Institute, Rockefeller University, New York, New York, USA.
Nat Struct Mol Biol. 2014 Aug;21(8):664-70. doi: 10.1038/nsmb.2851. Epub 2014 Jul 6.
Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.
真核生物使用不同的聚合酶进行前导链和滞后链复制,但它们如何靶向各自的链尚不清楚。我们重建了酿酒酵母复制叉,发现 CMG 解旋酶选择聚合酶 (Pol) ɛ 排除 Pol δ 在前导链上。即使 Pol δ 在前导链上组装,Pol ɛ 也会迅速取代它。Pol δ-PCNA 与 CMG 分布,与在引发 ssDNA 上的高稳定性形成对比。因此,CMG 不会稳定 Pol δ,而是使前导链可供 Pol ɛ 使用并稳定 Pol ɛ。在滞后链模型 DNA 上对 Pol ɛ 和 Pol δ 的比较显示出相反的情况。Pol δ 在 PCNA 引发的 ssDNA 上胜过多余的 Pol ɛ。因此,PCNA 在前导链上强烈偏向 Pol δ 而不是 Pol ɛ,但 CMG 推翻并翻转了这种平衡,在前导链上偏向 Pol ɛ。