| Autor: |
Alisa E. Lisova, Andrey G. Baranovskiy, Lucia M. Morstadt, Nigar D. Babayeva, Elena I. Stepchenkova, Tahir H. Tahirov |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
Scientific Reports, Vol 12, Iss 1, Pp 1-10 (2022) |
| Druh dokumentu: |
article |
| ISSN: |
2045-2322 |
| DOI: |
10.1038/s41598-022-21550-4 |
| Popis: |
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 (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 show that human PolεCD (hPolεCD) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolεCD produced in insect cells contains significantly lower level of iron. The iron content of purified hPolECD 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 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 K D of 105 µM. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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