Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland
Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
Life Sci Alliance. 2019 Jul 5;2(4). doi: 10.26508/lsa.201900321. Print 2019 Aug.
Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron-sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data hence suggest that the FeS cluster in human Pol δ is an important co-factor that despite its C-terminal location has an impact on both DNA polymerase and exonuclease activities, and can influence the fidelity of DNA synthesis.
真核生物基因组的复制依赖于 B 族 DNA 聚合酶 Pol α、Pol δ 和 Pol ε。这些酶都能协调一个铁硫（FeS）簇，但该辅因子的功能在很大程度上仍不清楚。在这里，我们表明人 Pol δ 的催化亚基中的 FeS 簇由 C 末端 CysB 基序的四个不变半胱氨酸配位。FeS 簇的丢失导致四亚基酶的部分不稳定，双链 DNA 结合缺陷，以及聚合酶和外切核酸酶活性受损。重要的是，在增殖细胞核抗原存在的情况下，复合物稳定性、DNA 结合和酶活性得到恢复。我们进一步表明，对 FeS 簇结合口袋的更细微的改变，即使不破坏 FeS 簇结合，也会对遥远的外切核酸酶结构域产生影响，使酶容易出错。因此，我们的数据表明，人 Pol δ 中的 FeS 簇是一个重要的辅因子，尽管它位于 C 末端，但对 DNA 聚合酶和外切核酸酶活性都有影响，并能影响 DNA 合成的准确性。
