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Published October 6, 2021 | Published + Supplemental Material
Journal Article Open

The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling

Abstract

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2_(CORE), are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2_(CORE)exo–, was used to limit DNA degradation. While no redox signal is apparent for Pol2_(CORE)exo– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2_(CORE)exo–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]²⁺, to oxidized Pol2_(CORE)exo– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIII^(Y82A), a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2_(CORE)exo–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2_(CORE)exo– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

Additional Information

© 2021 The Authors. Published by American Chemical Society. Attribution 4.0 International (CC BY 4.0). Received: July 9, 2021; Published: September 24, 2021. We are grateful to the Swedish Cancer Foundation (E.J.), Swedish Research Council (E.J.), Tryggers Stiftelse (E.J.), and NIH (Grant NIH GM126904 (J.K.B.)) for their financial support. We thank Prof. Jason Slinker for fabrication of multiplexed chips. We also thank Dr. Rebekah Silva and Dr. Adela Nano for help with protein assays, instrumentation, and valuable discussions. The authors declare no competing financial interest.

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Published - jacs.1c07150.pdf

Supplemental Material - ja1c07150_si_001.pdf

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Additional details

Created:
August 20, 2023
Modified:
October 23, 2023