Structure/Function Analysis of the Saccharomyces cerevisiae Trf4/Pol σ DNA Polymerase

Abstract

Abstract The Trf4p/Pol σ DNA polymerase (formerly Trf4p/Pol κ) couples DNA replication to the establishment of sister chromatid cohesion. The polymerase is encoded by two redundant homologs in Saccharomyces cerevisiae, TRF4 and TRF5, that together define a fourth essential nuclear DNA polymerase in yeast and probably in all eukaryotes. Here we present a thorough genetic analysis of the founding member of this novel family of DNA polymerases, TRF4. Analyses of mutants carrying 1 of 34 “surface-targeted” alanine scanning mutations in TRF4 have identified those regions required for Pol σ's essential function, for its role in DNA double-strand break repair, and for its association with chromosomes. The data strongly support the importance of the regions of predicted structural similarity with the Pol β superfamily as critical for Trf4p/Pol σ's essential and repair functions. Surprisingly, five lethal mutations lie outside all polymerase homology in a C-terminal region. The protein possesses Mg2+-dependent 3′ to 5′ exonuclease activity. Cell cycle analysis reveals that Trf4p/Pol σ associates with chromosomes in G1, S, and G2 phases, but that association is abolished coincident with dissolution of cohesion at the metaphase-to-anaphase transition.