ABSTRACT
The structure of the multisubunit yeast DNA polymerase epsilon (Pol epsilon) was determined to 20-A resolution using cryo-EM and single-particle image analysis. A globular domain comprising the catalytic Pol2 subunit is flexibly connected to an extended structure formed by subunits Dpb2, Dpb3 and Dpb4. Consistent with the reported involvement of the latter in interaction with nucleic acids, the Dpb portion of the structure directly faces a single cleft in the Pol2 subunit that seems wide enough to accommodate double-stranded DNA. Primer-extension experiments reveal that Pol epsilon processivity requires a minimum length of primer-template duplex that corresponds to the dimensions of the extended Dpb structure. Together, these observations suggest a mechanism for interaction of Pol epsilon with DNA that might explain how the structure of the enzyme contributes to its intrinsic processivity.
Subject(s)
DNA Polymerase II/chemistry , DNA Polymerase II/ultrastructure , Saccharomyces cerevisiae/enzymology , Catalysis , Cryoelectron Microscopy , DEAD-box RNA Helicases , DNA Polymerase II/metabolism , DNA, Fungal/chemistry , DNA, Fungal/metabolism , DNA, Fungal/ultrastructure , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA Helicases/chemistry , RNA Helicases/metabolism , RNA Helicases/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructureABSTRACT
De novo synthesis of fatty acids in the cytosol of animal cells is carried out by the multifunctional, homodimeric fatty acid synthase (FAS). Cryo-EM analysis of single FAS particles imaged under conditions that limit conformational variability, combined with gold labeling of the N termini and structural analysis of the FAS monomers, reveals two coiled monomers in an overlapping arrangement. Comparison of dimeric FAS structures related to different steps in the fatty acid synthesis process indicates that only limited local rearrangements are required for catalytic interaction among different functional domains. Monomer coiling probably contributes to FAS efficiency and provides a structural explanation for the reported activity of a FAS monomer dimerized to a catalytically inactive partner. The new FAS structure provides a new paradigm for understanding the architecture of FAS and the related modular polyketide synthases.