Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy

Department of Structural Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 02/2006; 13(1):35-43. DOI: 10.1038/nsmb1040
Source: PubMed


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.

Download full-text


Available from: Erik Johansson,
19 Reads
  • Source
    • "Pol a-B is arranged as an elongated structure organized in two lobes connected by a flexible linker. This quaternary organization has been observed for other eukaryotic replicative polymerases such as DNA polymerase ε (Asturias et al., 2006) and DNA polymerase d (Jain et al., 2009), which are responsible for the replication of the leading and lagging strands, respectively. In all these replicative complexes, the flexibility between the different lobes has been identified as a functional characteristic that allows the accommodation of different conformations of DNA during its replication and facilitates the interactions of these complexes with other components of the replication machinery. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This article presents a method to study large-scale conformational changes by combining electron microscopy (EM) single-particle image analysis and normal mode analysis (NMA). It is referred to as HEMNMA, which stands for hybrid electron microscopy normal mode analysis. NMA of a reference structure (atomic-resolution structure or EM volume) is used to predict possible motions that are then confronted with EM images within an automatic iterative elastic 3D-to-2D alignment procedure to identify actual motions in the imaged samples. HEMNMA can be used to extensively analyze the conformational changes and may be used in combination with classic discrete procedures. The identified conformations allow modeling of deformation pathways compatible with the experimental data. HEMNMA was tested with synthetic and experimental data sets of E. coli 70S ribosome, DNA polymerase Pol α and B subunit complex of the eukaryotic primosome, and tomato bushy stunt virus.
    Structure 02/2014; 22(3). DOI:10.1016/j.str.2014.01.004 · 5.62 Impact Factor
  • Source
    • "Consequently, one could envision Dpb3 and Dpb4 along with Dpb2 acting like a processivity factor, which would explain the unusual intrinsic high processivity of Pol e in the absence of PCNA. The presence of Dpb3/Dpb4 also plays a structural role in Pol e by stabilizing the conformation of Dpb2 (Asturias et al. 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Synopsis The current model of the eukaryotic DNA replication fork includes three replicative DNA poly-merases, polymerase a/primase complex (Pol a), polymerase d (Pol d), and polymerase e (Pol e). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymeriza-tion activity of Pol a into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol d utilizes PCNA to mediate highly processive DNA synthesis, while Pol e has intrinsic high processivity that is modestly stimulated by PCNA. Pol e replicates the leading strand and Pol d replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N-and C-terminal regions. The sizes of these inserts vary among the three poly-merases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol a, Pol d, and Pol e are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.
  • Source
    • "Indeed, this intermodular flexibility appears to be a general property of replicative B-family polymerases. An EM 3D model of the yeast Polε holoenzyme suggests analogous flexibility between the catalytic subunit (Pol2) and the accessory subunits (Dpb2, Dpb3, and Dpb4) (Asturias et al., 2006). Also, small-angle X-ray scattering analysis of the yeast Pold holoenzyme, which shares the Pol31-Pol32 subunits with Polz, suggests high conformational variability of the regulatory module with respect to the catalytic domain (Jain et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.
    Cell Reports 10/2013; 5(1). DOI:10.1016/j.celrep.2013.08.046 · 8.36 Impact Factor
Show more