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

YS Lee, MT Gregory, W Yang - Proceedings of the National …, 2014 - National Acad Sciences
YS Lee, MT Gregory, W Yang
Proceedings of the National Academy of Sciences, 2014National Acad Sciences
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 …
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.
National Acad Sciences