A Four-Subunit DNA Polymerase ζ Complex Containing Pol δ Accessory Subunits Is Essential for PCNA-mediated Mutagenesis

Nucleic Acids Res. 2012 Dec;40(22):11618-26. doi: 10.1093/nar/gks948. Epub 2012 Oct 12.

Abstract

DNA polymerase ζ (Pol ζ) plays a key role in DNA translesion synthesis (TLS) and mutagenesis in eukaryotes. Previously, a two-subunit Rev3-Rev7 complex had been identified as the minimal assembly required for catalytic activity in vitro. Herein, we show that Saccharomyces cerevisiae Pol ζ binds to the Pol31 and Pol32 subunits of Pol δ, forming a four-subunit Pol ζ(4) complex (Rev3-Rev7-Pol31-Pol32). A [4Fe-4S] cluster in Rev3 is essential for the formation of Pol ζ(4) and damage-induced mutagenesis. Pol32 is indispensible for complex formation, providing an explanation for the long-standing observation that pol32Δ strains are defective for mutagenesis. The Pol31 and Pol32 subunits are also required for proliferating cell nuclear antigen (PCNA)-dependent TLS by Pol ζ as Pol ζ(2) lacks functional interactions with PCNA. Mutation of the C-terminal PCNA-interaction motif in Pol32 attenuates PCNA-dependent TLS in vitro and mutagenesis in vivo. Furthermore, a mutant form of PCNA, encoded by the mutagenesis-defective pol30-113 mutant, fails to stimulate Pol ζ(4) activity, providing an explanation for the observed mutagenesis phenotype. A stable Pol ζ(4) complex can be identified in all phases of the cell cycle suggesting that this complex is not regulated at the level of protein interactions between Rev3-Rev7 and Pol31-Pol32.

Publication types

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

MeSH terms

  • Cell Cycle
  • DNA Polymerase III / metabolism*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism*
  • Iron-Sulfur Proteins
  • Mutagenesis*
  • Mutation
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Structure, Tertiary
  • Protein Subunits / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Iron-Sulfur Proteins
  • Pol32 protein, S cerevisiae
  • Proliferating Cell Nuclear Antigen
  • Protein Subunits
  • REV7 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • DNA Polymerase III
  • DNA polymerase zeta
  • DNA-Directed DNA Polymerase
  • POL31 protein, S cerevisiae
  • REV3 protein, S cerevisiae