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An iron-sulfur cluster in the polymerase domain of yeast DNA polymerase ε
Rinku Jain1, Eva S Vanamee1, Boris G Dzikovski2
1Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA.
Abstract:
DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.
Insights
DNA polymerase ε (Polε), crucial for DNA replication and repair, contains an iron-sulfur [Fe-S] cluster in its active site. This cluster is essential for Polε
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- DNA polymerase ε (Polε) is vital for maintaining genomic stability through leading strand replication and DNA repair.
- Polε in Saccharomyces cerevisiae comprises four subunits: Pol2, Dpb2, Dpb3, and Dpb4.
Purpose of the Study:
- To investigate the structural and functional role of a newly identified iron-sulfur [Fe-S] cluster within the Polε active site.
- To determine the impact of the [Fe-S] cluster on Polε's polymerase and exonuclease activities.
Main Methods:
- Biochemical assays to characterize the [Fe-S] cluster binding site within the Pol2 subunit.
- Enzyme activity assays to assess the necessity of the [Fe-S] cluster for polymerase and exonuclease functions.
Main Results:
- An [Fe-S] cluster is located within the active polymerase domain of the Pol2 subunit (residues 1-1187).
- The [Fe-S] cluster binding is mediated by conserved cysteines in a Pol2-specific insertion (Pol2(ins)).
- The [Fe-S] cluster is indispensable for Pol2 polymerase activity but not its exonuclease activity.
Conclusions:
- The [Fe-S] cluster is a critical component of DNA polymerase ε's catalytic activity.
- Polε's unique [Fe-S] cluster suggests a potential heightened sensitivity to oxidative stress compared to other DNA polymerases.
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