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Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase epsilon
- Source :
- 'Nucleic Acids Research ', vol: 47, pages: 5712-5722 (2019), Nucleic Acids Research
- Publication Year :
- 2019
-
Abstract
- DNA polymerase epsilon (Pol epsilon), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2(CORE)) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2(CORE) (with the CysX cysteine motif) are likely to coordinate an Fe-S cluster. Here, we present two new crystal structures of Pol2(CORE) with an Fe-S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol epsilon, Pol epsilon CysA(MUT) (C2111S/C2133S), and Pol epsilon CysB(MUT) (C2167S/C2181S) all have an Fe-S cluster that is not present in Pol epsilon CysX(MUT) (C665S/C668S). Pol epsilon CysA(MUT) and Pol epsilon CysB(MUT) behave similarly to wildtype Pol epsilon in in vitro assays, but Pol epsilon CysX(MUT) has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysX(MUT) are inviable. In conclusion, Pol epsilon has a single Fe-S cluster bound at the base of the P-domain, and this Fe-S cluster is essential for cell viability and polymerase activity.
- Subjects :
- DNA Replication
Iron-Sulfur Proteins
Exonuclease
congenital, hereditary, and neonatal diseases and abnormalities
Saccharomyces cerevisiae Proteins
DNA polymerase
Stereochemistry
Protein subunit
viruses
Amino Acid Motifs
Protein domain
DNA-Directed DNA Polymerase
Saccharomyces cerevisiae
Genome Integrity, Repair and Replication
Crystallography, X-Ray
DNA-binding protein
Genome
03 medical and health sciences
0302 clinical medicine
Protein Domains
Catalytic Domain
Genetics
Humans
Cysteine
030304 developmental biology
0303 health sciences
biology
DNA replication
Biochemistry and Molecular Biology
DNA Polymerase II
biology.protein
Genome, Fungal
Oxidation-Reduction
030217 neurology & neurosurgery
Biokemi och molekylärbiologi
Protein Binding
Subjects
Details
- Language :
- English
- ISSN :
- 03051048
- Volume :
- 47
- Database :
- OpenAIRE
- Journal :
- Nucleic Acids Research
- Accession number :
- edsair.doi.dedup.....74e702e251deca519c4a8ffa76dfbd3f