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 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.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Young-Sam
YS
Lee
Mark T
MT
Gregory
Wei
W
Yang
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002945:Cisplatin / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis*; D004258:DNA Polymerase III / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D019793:Fluorescein; D057809:HEK293 Cells; D020035:Holoenzymes / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D064246:Mad2 Proteins / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D002945:Cisplatin; D004247:DNA; D004258:DNA Polymerase III; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D019793:Fluorescein; D020035:Holoenzymes; D064246:Mad2 Proteins; C120552:MAD2L2 protein, human; C425929:DNA polymerase zeta; C435720:POLD3 protein, human; C118285:REV3L protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
MAD2L2; REV3L; TLS; processivity; two-polymerase lesion bypass