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结构证据表明 DNA 聚合酶 ϵ 的催化核心结构域中存在必需的 Fe-S 簇。

Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase ϵ.

机构信息

Department of Medical Biochemistry and Biophysics, Umeå University, Umeå 90187, Sweden.

Department of Chemistry, Umeå University, Umeå, 90187, Sweden.

出版信息

Nucleic Acids Res. 2019 Jun 20;47(11):5712-5722. doi: 10.1093/nar/gkz248.

DOI:10.1093/nar/gkz248
PMID:30968138
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6582351/
Abstract

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe-S cluster. Here, we present two new crystal structures of Pol2CORE with an Fe-S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe-S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT are inviable. In conclusion, Pol ϵ has a single Fe-S cluster bound at the base of the P-domain, and this Fe-S cluster is essential for cell viability and polymerase activity.

摘要

DNA 聚合酶 ϵ（Pol ϵ）是真核生物中主要的领头链 DNA 聚合酶，它有一个催化亚基（Pol2）和三个非催化亚基。Pol2 的 N 端半区（Pol2CORE）具有聚合酶和外切核酸酶活性。有人提出，Pol2 的非催化 C 端结构域（带有两个半胱氨酸基序 CysA 和 CysB）和 Pol2CORE（带有 CysX 半胱氨酸基序）可能都能协调一个 Fe-S 簇。在这里，我们提出了两个新的晶体结构，其中 Pol2CORE 与 CysX 基序结合了一个 Fe-S 簇，该结构得到了该位置的异常信号的支持。此外，我们还表明，纯化的四聚体 Pol ϵ、Pol ϵ CysAMUT（C2111S/C2133S）和 Pol ϵ CysBMUT（C2167S/C2181S）都有一个 Fe-S 簇，而 Pol ϵ CysXMUT（C665S/C668S）中则没有。Pol ϵ CysAMUT 和 Pol ϵ CysBMUT 在体外实验中与野生型 Pol ϵ 表现相似，但 Pol ϵ CysXMUT 的 DNA 聚合酶活性严重受损，这不是外切核酸酶活性过度的结果。四联体分析表明，携带 CysXMUT 的单倍体酵母菌株是不可存活的。总之，Pol ϵ 在 P 结构域的底部有一个单一的 Fe-S 簇，这个 Fe-S 簇对于细胞活力和聚合酶活性是必不可少的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/30d72bd71f12/gkz248fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/299383ef9f6a/gkz248fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/4c7da2a20f60/gkz248fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/92b694a8d6b2/gkz248fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/4cca108383d7/gkz248fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/6cec0a6f4fdd/gkz248fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/1880812d9549/gkz248fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/a27cfe45854f/gkz248fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/30d72bd71f12/gkz248fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/299383ef9f6a/gkz248fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/4c7da2a20f60/gkz248fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/92b694a8d6b2/gkz248fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/4cca108383d7/gkz248fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/6cec0a6f4fdd/gkz248fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/1880812d9549/gkz248fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/a27cfe45854f/gkz248fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d8b2/6582351/30d72bd71f12/gkz248fig8.jpg

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