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Plasmids and Strains. To create pNTAP-Swi1, the swi1 cDNA ORF was PCR-amplified by overlap PCR to eliminate the sole intron and cloned into pREP1NTAP (provided by K L. Gould, Vanderbilt University, Nashville, TN) as an NdeI–SmaI fragment. To generate pcGST-Swi1 WT, the swi1 cDNA ORF was PCR-amplified from pNTAP-Swi1 and cloned into pRL3 (provided by R. Dhar, National Institutes of Health, Bethesda) as a BamHI–SmaI fragment. pcGST-Swi1 E662K was created in a similar manner by overlap PCR with DNA derived from JZ277 as template. pIS8B.IRT2 consists of 252 bp upstream of and containing the rDNA intergenic HindIII site, cloned as a BamHI fragment into pIRT2. To create pTer2.IRT2 and p1REB1.IRT2, a 250-bp fragment and a 257-bp fragment containing the downstream and upstream Reb1p-binding elements (with respect to rRNA transcription) and corresponding to Ter2 and Ter3, respectively, were cloned into pIRT2 as BamHI fragments. pIS8B.IRT2, pTer2.IRT2, and p1REB1.IRT2 contain Ter1, Ter2, and Ter3, respectively, in their native orientations with respect to ars1 of pIRT2. To create pRFP4b.IRT2, ≈400 bp of DNA sequence immediately upstream of the farthest upstream Reb1p binding site Ter3 (with respect to transcription) was cloned in its native orientation as a BamHI fragment into the pIRT2 shuttle vector. pRFP4a.IRT2 was created in a similar manner but used ≈1 kb of immediate upstream sequence.

SPGK100 (h⁹⁰, ade6-M210, ura4-D18, leu1-32, reb1D ::kanMX) was derived from SP976 by using a three-piece PCR strategy and standard gene-replacement techniques (1). Briefly, regions immediately upstream and downstream of the reb1 ORF were PCR-amplified, gel-purified (Qiagen, Valencia, CA), and subsequently used as primers to PCR the kanMX G418 resistance gene from p2021 (provided by K. L. Gould). This third PCR product was used to transform SP976. G418-resistant colonies were picked and restreaked. Gene replacement was verified by PCR, sequencing, and Southern blotting. SPGK301C (ade6-M210, ura4-D18, leu1-32, swi1::ura4) are also derived from SP976. Briefly, the swi1 ORF was cloned into pUC18 at the unique SmaI site and digested with AfeI. Subsequently, ura4+ was PCR-amplified from pDW232 (Invitrogen) and blunt-end ligated to the disrupted swi1 ORF, thereby generating pswi1::ura4. This plasmid was then linearized at the unique BsrFI site and transformed into SP976. Ura4+ colonies were analyzed for defective mating-type switching and gene replacement was subsequently verified by PCR. Sequences of all primers used for plasmid and strain construction are available upon request.

Preparation of Plasmid and Chromosomal DNA for Neutral/Neutral 2D Agarose Gel Electrophoresis. To capture replication intermediates for neutral/neutral 2D gels, actively growing cells were killed with 0.1% sodium azide and immediately chilled on ice/glycerol/EDTA and harvested. Cell pellets were washed once with chilled water and frozen at –80°C until use. DNA was isolated essentially as described by Huberman et al. (2) with modifications. Briefly, cells were resuspended in nuclear isolation buffer at a density of ≈2 × 10⁹/ml and lysed by using acid-washed glass beads (Sigma). Nuclei were pelleted in a Sorvall SS34 at 8,000 rpm for 10 min and resuspended in 50 mM Tris (pH 8.0)/50 mM EDTA/100 mM NaCl containing 0.1% SDS and 300 m g/ml Proteinase K (Invitrogen). After incubation for 1 h at 37°C, SDS was adjusted to 1%, and incubation was continued at 37°C for 1 h, followed by 4°C for 2–3 h. Potassium acetate was added to 1.1 M for 1 h at 4°C, and the debris was spun out for 10 min at 10,000 rpm. An equal volume of isopropanol was added to the supernatant, and the precipitated nucleic acids were centrifuged for 10 min at 10,000 rpm. Dried pellets were dissolved in 10 mM Tris/1 mM EDTA (pH 8.0) overnight and subsequently extracted once with phenol/chloroform/isoamyl alcohol (25:24:1) and once with choloroform/isoamyl alcohol (24:1). After ethanol precipitation, the pellets were again dissolved in 10 mM Tris/1 mM EDTA (pH 8.0) and RNase A (20 m g/ml), and the appropriate restriction enzyme(s) were added for 5–6 h at 37°C. Neutral/neutral 2D gels were performed as described in refs. 2 and 3. First-dimension electrophoresis was performed in 0.6% agarose and the second-dimension in 1.0% or 1.2%.

1. Moreno, S., Klar, A. & Nurse, P. (1991) Methods Enzymol. 194, 795–823.

2. Huberman, J. A., Spotila, L. D., Nawotka, K. A., el-Assouli, S. M. & Davis, L. R. (1987) Cell 51, 473–481.

3. Brewer, B. J., Lockshon, D. & Fangman, W. (1992) Cell 71, 267–271.