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

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dc.contributor.authorLisova, A.E.
dc.contributor.authorBaranovskiy, A.G.
dc.contributor.authorMorstadt, L.M.
dc.contributor.authorBabayeva, N.D.
dc.contributor.authorStepchenkova, E.I.
dc.contributor.authorTahirov, T.H.
dc.date.issued2022
dc.description.abstractDNA 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 KD of 105 μM.
dc.description.statementofresponsibilityAlisa E. Lisova, Andrey G. Baranovskiy, Lucia M. Morstadt, Nigar D. Babayeva, Elena I. Stepchenkova, Tahir H. Tahirov
dc.identifier.citationScientific Reports, 2022; 12(1):17436-1-17436-10
dc.identifier.doi10.1038/s41598-022-21550-4
dc.identifier.issn2045-2322
dc.identifier.issn2045-2322
dc.identifier.orcidLisova, A.E. [0000-0002-3647-6460]
dc.identifier.urihttps://hdl.handle.net/2440/146467
dc.language.isoen
dc.publisherNature Portfolio
dc.rights© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
dc.source.urihttps://doi.org/10.1038/s41598-022-21550-4
dc.subjectDNA; Cysteine; Exonucleases; Iron-Sulfur Proteins; Saccharomyces cerevisiae; Biochemistry; Structural biology
dc.subject.meshHumans
dc.subject.meshSaccharomyces cerevisiae
dc.subject.meshIron
dc.subject.meshCysteine
dc.subject.meshExonucleases
dc.subject.meshDNA Polymerase II
dc.subject.meshIron-Sulfur Proteins
dc.subject.meshDNA
dc.subject.meshKinetics
dc.titleThe iron-sulfur cluster is essential for DNA binding by human DNA polymerase ε.
dc.typeJournal article
pubs.publication-statusPublished

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