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

Proc Natl Acad Sci U S A. 2014 Feb 25;111(8):2954-9. doi: 10.1073/pnas.1324001111. Epub 2014 Jan 21.

Abstract

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.

Keywords: MAD2L2; REV3L; TLS; processivity; two-polymerase lesion bypass.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cisplatin / metabolism*
  • 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
  • HEK293 Cells
  • Holoenzymes / isolation & purification
  • Holoenzymes / metabolism
  • Humans
  • Mad2 Proteins / metabolism

Substances

  • DNA Primers
  • DNA-Binding Proteins
  • Holoenzymes
  • MAD2L2 protein, human
  • Mad2 Proteins
  • DNA
  • DNA polymerase zeta
  • POLD3 protein, human
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase
  • REV3L protein, human
  • Rad30 protein
  • Cisplatin
  • Fluorescein