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.

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    • "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. "
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    • "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). "
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    • "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). "
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