A four-subunit DNA polymerase ? complex containing Pol ? accessory subunits is essential for PCNA-mediated mutagenesis.

ABSTRACT: DNA polymerase ? (Pol ?) 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 ? binds to the Pol31 and Pol32 subunits of Pol ?, forming a four-subunit Pol ?(4) complex (Rev3-Rev7-Pol31-Pol32). A [4Fe-4S] cluster in Rev3 is essential for the formation of Pol ?(4) and damage-induced mutagenesis. Pol32 is indispensible for complex formation, providing an explanation for the long-standing observation that pol32? strains are defective for mutagenesis. The Pol31 and Pol32 subunits are also required for proliferating cell nuclear antigen (PCNA)-dependent TLS by Pol ? as Pol ?(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 ?(4) activity, providing an explanation for the observed mutagenesis phenotype. A stable Pol ?(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.

SUBMITTER: Makarova AV 

PROVIDER: S-EPMC3526297 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature