Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA.
DNA Replication Laboratory and Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA.
Nat Commun. 2024 Sep 8;15(1):7847. doi: 10.1038/s41467-024-52257-x.
In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Polε catalytic domain has been determined, how Polε interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Polε-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Polε catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Polε. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding.
在真核生物中，先导链 DNA 由 Polε 合成，滞后链由 Polδ 合成。这些复制聚合酶与 DNA 夹 PCNA 结合时具有更高的持续性。虽然已经确定了酵母 Polε 催化结构域的结构，但在任何真核生物（人类或酵母）中，Polε 与 PCNA 的相互作用方式尚不清楚。在这里，我们报告了人类 Polε-PCNA-DNA 复合物的两个冷冻电镜结构，一个处于进入核苷酸结合状态，另一个处于核苷酸交换状态。这些结构揭示了 Polε 催化结构域与 PCNA 之间一个出乎意料的三点界面，其中保守的 PIP（PCNA 相互作用肽）基序、独特的 P 结构域和拇指结构域分别与 PCNA 三聚体的不同亚基相互作用。我们提出，多点界面可防止其他含有 PIP 的因子在 PCNA 与 Polε 一起发挥作用时募集到 PCNA。两个状态的比较表明，指结构域围绕 P 结构域含[4Fe-4S]簇的尖端枢转，以调节核苷酸交换和进入核苷酸结合。
