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DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. It is attached to a helicase and performs DNA synthesis on the leading strand. Recently it was shown that the catalytic domain of yeast Polε (PolεCD) 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 have shown that human PolεCD (hPolεCD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolεCD produced in insect cells contains an eight-fold-lower level of iron. Interestingly, the iron content 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 and, therefore, 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. In addition, we performed kinetic and binding studies of hPolεCD. The binding studies demonstrated low affinity of hPolεCD to DNA and a strong effect of salt concentration on stability of the hPolεCD/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 KD of 105 μM. This work provides notable insight into the role of a [4Fe-4S] cluster in Polε function. |
