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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_COREexo^-, was used to limit DNA degradation. While no redox signal is apparent for Pol2_COREexo^- 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_COREexo^-, 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_COREexo^- 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_COREexo^-. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2_COREexo^- via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

SUBMITTER: Pinto MN

PROVIDER: S-EPMC8499023 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Comment on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport".

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Response to Comments on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport".

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