Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as lar...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2014-02, Vol.111 (8), p.2954-2959
Main Authors: Lee, Young-Sam, Gregory, Mark T., Yang, Wei
Format: Article
Language:English
Subjects:
Biological Sciences
Catalytic activity
Chromatography, Gel
Chromatography, Ion Exchange
Cisplatin - metabolism
DNA
DNA - biosynthesis
DNA Polymerase III - metabolism
DNA Primers - genetics
DNA Repair - physiology
DNA-Binding Proteins - metabolism
DNA-Directed DNA Polymerase - isolation & purification
DNA-Directed DNA Polymerase - metabolism
Fluorescein
Genetic mutation
HEK293 Cells
Holoenzymes - isolation & purification
Holoenzymes - metabolism
Humans
Lesions
Mad2 Proteins - metabolism
Nucleic acids
Nucleotides
Proteins
Saccharomyces cerevisiae
Yeasts
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Summary:DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified four-subunit Pol ζ4 (Rev3–Rev7–PolD2–PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3–Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3′ guanine and Pol ζ4 to extend the primers.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1324001111