Reference - PMID:36261579 - The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε.
Reference summary
- PubMed ID
- PMID:36261579
- Title
- The iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε.
- Authors
- Lisova AE, Baranovskiy AG, Morstadt LM, Babayeva ND, Stepchenkova EI, Tahirov TH
- Citation
- Sci Rep 2022 Oct 19;12(1):17436
- Publication year
- 2022
- Abstract
- 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 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 Polε CD (hPolε CD ) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolε 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 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 CD to DNA and a strong effect of salt concentration on stability of the hPolε 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 µM.
