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Published November 6, 1998 | Published
Journal Article Open

Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase epsilon in DNA Replication and the S/M Checkpoint Pathway


It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) epsilon (POL2) couple DNA replication to the S/M checkpoint (Navas, T. A., Zheng, Z., and Elledge, S. J. (1995) Cell 80, 29-39). Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint. All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response. The mutations affect the assembly of the pol epsilon holoenzyme. Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol epsilon . The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol epsilon , but not for interaction with DPB3. Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay.

Additional Information

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, June 8, 1998, and in revised form, August 26, 1998) We thank the members of the Campbell laboratory for helpful discussions and Dr. Julie Archer for help in immunofluorescence studies. We are grateful to Akio Sugino and Hiroyuki Araki for pol epsilon antibodies and the pol2 knock-out strain, YHA301. This work was supported by PHS grant GM25508 and American Heart Association Research Fellowship Award 1153-F12.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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