Analysis of the essential functions of the C-terminal protein/protein interaction domain of Saccharomyces cerevisiae pol epsilon and its unexpected ability to support growth in the absence of the DNA polymerase domain.
As first observed by Wittenberg (Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenburg, C. (1999) Mol. Cell 3, 679-685), we find that deletion mutants lacking the entire N-terminal DNA polymerase domain of yeast pol epsilon are viable. However, we now show that point mutations in DNA polymerase catalytic residues of pol epsilon are lethal. Taken together, the phenotypes of the deletion and the point mutants suggest that the polymerase of pol epsilon may normally participate in DNA replication but that another polymerase can substitute in its complete absence. Substitution is inefficient because the deletion mutants have serious defects in DNA replication. This observation raises the question of what is the essential function of the C-terminal half of pol epsilon. We show that the ability of the C-terminal half of the polymerase to support growth is disrupted by mutations in the cysteine-rich region, which disrupts both dimerization of the POL2 gene product and interaction with the essential DPB2 subunit, suggesting that this region plays an important architectural role at the replication fork even in the absence of the polymerase function. Finally, the S phase checkpoint, with respect to both induction of RNR3 transcription and cell cycle arrest, is intact in cells where replication is supported only by the C-terminal half of pol epsilon, but it is disrupted in mutants affecting the cysteine-rich region, suggesting that this domain directly affects the checkpoint rather than acting through the N-terminal polymerase active site.
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Braun Laboratories 147-75, California Institute of Technology, Pasadena, California 91125, USA.
Levy
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DL
Campbell
J L
JL
D020134:Catalytic Domain; D003545:Cysteine / Q000235:genetics; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D019281:Dimerization; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D016335:Zinc Fingers
D004257:DNA Polymerase II; D003545:Cysteine