A four-subunit DNA polymerase ζ complex containing Pol δ accessory subunits is essential for PCNA-mediated mutagenesis.
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.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Alena V
AV
Makarova
Joseph L
JL
Stodola
Peter M
PM
Burgers
D002453:Cell Cycle; D004258:DNA Polymerase III / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins; D016296:Mutagenesis; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; C522758:Pol32 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; C092219:REV7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C425929:DNA polymerase zeta; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C095943:POL31 protein, S cerevisiae; C092602:REV3 protein, S cerevisiae