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Published September 15, 2000 | Published
Journal Article Open

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase ε (pol ε) complex - Demonstration of a dimeric pol ε


Saccharomyces cerevisiae DNA polymerase epsilon (pol ε) is essential for chromosomal replication. A major form of pol ε purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2pzDpb2p and Dpb3pzDpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol ε may be dimeric in vivo. Gel filtration of the Pol2pzDpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2pzDpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.

Additional Information

© 2000 American Society for Biochemistry and Molecular Biology. Received for publication, March 20, 2000, and in revised form, June 8, 2000. We are grateful to Akio Sugino and Hiroyuki Araki for communication of results prior to publication and to Akio Sugino for antibody to pol Є. This work was supported by United States Public Health Service Grant 25508 and Grant 1153-F12 from the American Heart Association (to R.D.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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