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Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes

Nature Chemical Biology volume 8, pages 125–132 (2012)Cite this article

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Abstract

The eukaryotic replicative DNA polymerases (Pol α, δ and ɛ) and the major DNA mutagenesis enzyme Pol ζ contain two conserved cysteine-rich metal-binding motifs (CysA and CysB) in the C-terminal domain (CTD) of their catalytic subunits. Here we demonstrate by in vivo and in vitro approaches the presence of an essential [4Fe-4S] cluster in the CysB motif of all four yeast B-family DNA polymerases. Loss of the [4Fe-4S] cofactor by cysteine ligand mutagenesis in Pol3 destabilized the CTD and abrogated interaction with the Pol31 and Pol32 subunits. Reciprocally, overexpression of accessory subunits increased the amount of the CTD-bound Fe-S cluster. This implies an important physiological role of the Fe-S cluster in polymerase complex stabilization. Further, we demonstrate that the Zn-binding CysA motif is required for PCNA-mediated Pol δ processivity. Together, our findings show that the function of eukaryotic replicative DNA polymerases crucially depends on different metallocenters for accessory subunit recruitment and replisome stability.

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Figure 1: Yeast replicative DNA polymerases and Rev3 contain Fe-S clusters in vivo.
Figure 2: The Fe-S cluster is coordinated by the CysB motif and is stabilized by accessory-subunit binding.
Figure 3: Recombinant purified CTDs of Pol1, Pol2, Pol3 and Rev3 harbor a [4Fe-4S] cluster.
Figure 4: Functional integrity of purified Pol δ complex depends on binding of an Fe-S cluster to Pol3.
Figure 5: The CysA motif of Pol δ is critical for processive DNA replication with PCNA.
Figure 6: Role of mitochondria and Fe-S cluster biogenesis in nuclear DNA replication.

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Acknowledgements

We thank B. Yoder for yeast two-hybrid analysis and M. Reuter for help with cloning. This work was supported by grant GM032431 from the US National Institutes of Health (P.M.J.B.), the Deutsche Forschungsgemeinschaft (SFB 593, Gottfried-Wilhelm Leibniz program, and GRK 1216), Rhön Klinikum, von Behring-Röntgen Stiftung, LOEWE-Program of State Hessen, Max-Planck Gesellschaft and Fonds der Chemischen Industrie.

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  1. Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Marburg, Germany

    Daili J A Netz, Martin Stümpfig, Gabriele Köpf, Daniel Vogel, Heide M Genau, Roland Lill & Antonio J Pierik

  2. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA

    Carrie M Stith, Joseph L Stodola & Peter M J Burgers

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All authors performed experiments. D.J.A.N., R.L., P.M.J.B. and A.J.P. designed experiments, analyzed data and wrote the manuscript.

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Correspondence to Roland Lill, Peter M J Burgers or Antonio J Pierik.

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Netz, D., Stith, C., Stümpfig, M. et al. Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes. Nat Chem Biol 8, 125–132 (2012). https://doi.org/10.1038/nchembio.721

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