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Published November 1, 1983 | Published
Journal Article Open

Isolation of Yeast DNA Replication Mutants in Permeabilized Cells


A random population of temperature-sensitive mutants was screened by assaying for defects in DNA synthesis in a permeabilized yeast DNA replication system. Twenty mutants defective in in vitro DNA synthesis have been isolated. In this paper we describe eight of these mutants. Seven of them fall into three complementation groups--cdc2, cdc8, and cdc16--involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in vivo at the time of permeabilization, our finding that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23 degrees C but not at 37 degrees C supports the conclusion that these two mutations directly affect DNA synthesis at replication forks. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. Finally, the usefulness of our screening procedure is demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.

Additional Information

© 1983 by the National Academy of Sciences Communicated by Leroy Hood, July 15, 1983 We thank Joan Kobori, Caren Chang, and Ambrose Jong for helpful comments on the manuscript. This work was supported by grants from the U.S. Public Health Service (GM 25508 and T 32 GM 07616), the American Cancer Society (MV-142), and the March of Dimes (1-794). J.L.C. is the recipient of Research Career Development Award CA00544. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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