posted on 2021-09-24, 18:05authored byMiguel
N. Pinto, Josy ter Beek, Levi A. Ekanger, Erik Johansson, Jacqueline K. Barton
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,
Pol2CORE, are explored using DNA electrochemistry, and
the effects of oxidation and rereduction on polymerase activity are
examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo–, was used to limit DNA degradation.
While no redox signal is apparent for Pol2COREexo– 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 Pol2COREexo–, 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 Pol2COREexo– bound to its DNA
substrate also significantly restores polymerase activity. In contrast,
parallel experiments with EndoIIIY82A, a variant of EndoIII,
defective in DNA charge transport (CT), does not show restoration
of activity of Pol2COREexo–. We propose
a model in which EndoIII bound to the DNA duplex may shuttle electrons
through DNA to the DNA-bound oxidized Pol2COREexo– via DNA CT and that this DNA CT signaling offers a means to modulate
the redox state and replication by Pol ε.