The Polε catalytic subunit contains three cysteine motifs: CysA, CysB, and CysX 10, 11. Instability of hPolε holoenzyme results in replication stress, tumorigenesis, and developmental abnormalities 8, 9. In the recently reported structure of the yeast Polε holoenzyme, the small subunits p17 and p12 are tethering the two lobes of p261 7. The p261 is composed of two duplicated exonuclease/polymerase domains, the first of which is active, while the second is inactive and plays an important structural role 4, 5, 6. Human Polε (hPolε) consists of four subunits: the catalytic subunit (p261) and the accessory subunits p59, p17, and p12, listed in order from bigger to smaller. It belongs to the B-family of DNA polymerases, which also contains Polδ, Polα, and Polζ.
Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s −1 and a relatively low affinity to incoming dNTP with an apparent K D of 105 µM.ĭNA polymerase ε (Polε) is one of the main eukaryotic replicases responsible for leading-strand synthesis during genome duplication 1, 2, 3. The binding studies demonstrated low affinity of hPolε CD to DNA and a strong effect of salt concentration on stability of the hPolε CD/DNA complex. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding as well as DNA polymerase and proofreading exonuclease activities. The iron content of purified hPolE CD samples correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA.
In comparison, recombinant hPolε CD produced in insect cells contains significantly lower level of iron. In this work, we show that human Polε CD (hPolε CD) expressed in bacterial cells also contains an iron-sulfur cluster. Recently it was shown that the catalytic domain of yeast Polε (Polε CD) contains a cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes.
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