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

DNA polymerase epsilon (Pol epsilon) is a key enzyme for DNA replication in eukaryotes. Recently it was shown that the catalytic domain of yeast Pol epsilon (Pol epsilon(CD)) 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 Pol epsilon(CD) (hPol epsilon(CD)) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPol epsilon(CD) produced in insect cells contains significantly lower level of iron. 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 hPol epsilon 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 hPol epsilon(CD) to DNA and a strong effect of salt concentration on stability of the hPol epsilon(CD)/DNA complex. 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 mu M.

Automated summary

DNA polymerase epsilon plays a crucial role in DNA replication in eukaryotes and contains an iron-sulfur cluster that regulates enzyme conformation and interacts with DNA. Mutation of conserved cysteines coordinating the cluster abolishes DNA binding and polymerase activity.