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Cdc45p was tagged with three copies of the hemagglutinin (HA) epitope at its COOH-terminus. The strains generated (YB0469, MATa, cdc45:: CDC45-HA3-LEU2; YB0470, MATα, same as YB0469) grew identically to the parental strain (W303-1) at 25° and 37°C. Extract preparation, centrifugal fractionation, and MNase digestion were done essentially as described in (7), except that the extraction buffer contained 1% Triton X-100. Samples were resolved on 10% SDS-polyacrytamide gels. Cdc45-HA3p, Orc3p, Mcm2p, and Mcm3p were detected with monoclonal antibodies 12CA5 (HA antibody), SB3 (Orc3p antibody), Mcm239, and Mcm34, respectively.
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7) were collected at each time point. Low-speed supernatant and sedimented fractions were prepared as described in (7).
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The following Cdc45-HA3p - expressing mutants are in the W303 strain background. YB0471, cdc6-1; YB0472, cdc7-1; YB0473, cdc15-2; YB0474, cdc28-4; YB0475, cdc34; YB0476, mcm2; YB0477, Clb5::URA3, Clb6::LEU2. The following Cdc45-HA3p - expressmg strains are not in the W303 strain background: YB0478, cdc13; YB0479, cdc17-1; YB0480, dbf2-2. All these strains exhibit temperature sensitivity similar to the parental mutant strains.
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We thank S. Bell and M. Akiyama for antibodies, C. Liang for advice on chromatin fractionation, and J. Chong, A. Verreault, and H. Yang for critical reading of the manuscript. Supported by a grant from NIH , (GM45436 to B.S.).
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