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Plasmid and strain construction: All strains were congenic with Y300 except those derived from the strains provided by T. Weinert (TW) as indicated and were derived with standard genetic techniques (10). CHK1 (accession number AF117345) was disrupted by using the disruption cassette pYS51 (chk1::HIS3). PDS1 was disrupted by using pAY55 (13). The cdc14-1 allele was introduced into Y300 using the pSD231 cassette. The cdc5-1 allele was backcrossed into the Y300 background five times from an initial cross with strain 11359-10-4A provided by D. Toczyski and L. Hartwell. Strains containing deletions of MEC1 or RAD53 are maintained by expression of RNR1 under GAP control (26). The RAD9 and CHK1 ORFs were introduced into pACT-lox and pAS2-lox via the UPS reaction (27).
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note
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We thank A. Murray, A. Straight, T. Weinert, D. Gottschling, O. Cohen-Fix, D. Koshland, V. Lundblad, M. Yanagida, U. Surana, L. Hartwelt, D. Toczyski, and A. Amon for providing reagents and helpful information; C. Nugent, C. Tomlinson, and M. Kuroda for helpful comments on the manuscript; and D. Honican and M. Li for excellent technical assistance. Y.S. and J.B. were supported by NIH postdoctoral fellowships. Supported by grants GM44664 and Q1187 (Welch) to S.J.E. S.J.E is an investigator with the Howard Hughes Medical Institute.
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