The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε.

DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. Recently it was shown that the catalytic domain of yeast Polε (Polε) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we show that human Polε (hPolε) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolε produced in insect cells contains significantly lower level of iron. The iron content of purified hPolE samples correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding as well as DNA polymerase and proofreading exonuclease activities. 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. The binding studies demonstrated low affinity of hPolε to DNA and a strong effect of salt concentration on stability of the hPolε/DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s and a relatively low affinity to incoming dNTP with an apparent K of 105 µM.