TABLE 1.
Strains used in this study
| Strain | Genotype |
|---|---|
| RUY121 | Wild type; MATα ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 |
| RUY139 | MATα sic1∷HIS3 |
| RUY275 | MATα orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm |
| RUY384 | MAT? orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm sic1∷HIS3 |
| RUY385 | MAT? orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm sic1∷HIS3 clb5∷URA3 clb6∷KanMX |
| RUY386 | MATaorc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm sic1∷HIS3 clb6∷KanMX |
| RUY387 | MATα orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm clb5∷URA3 clb6∷KanMX |
| RUY140 | MATα cdh1∷LEU2 |
| RUY263 | MATα URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY227 | MATα clb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY174 | MATaclb5∷URA3 clb6∷KanMX URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY228 | MATaMCM7-NLS URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY179 | MATaMCM7-NLS clb5∷URA3 clb6∷KanMX URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY170 | MATα orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY181 | MATα orc6∷HIS3∷LEU2∷ORC6-ps,rxl clb5∷URA3URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY184 | MATaorc6∷HIS3∷LEU2∷ORC6-ps,rxl clb5∷URA3 clb6∷KanMX URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY169 | MATα MCM7-NLS orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY191 | MATaMCM7-NLS orc6∷HIS3∷LEU2∷ORC6-ps,rxl clb5∷URA3 clb6∷KanMX URA3∷GAL-CDC6ΔNT-HAm DDC2-GFP∷TRP1 |
| RUY325 | MATα MCM7-NLS clb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm |
| RUY326 | MATα clb5∷URA3 URA3∷ GAL-CDC6ΔNT-HAm MCM7-NLS cdh1∷LEU2 |
| RUY172 | MATaclb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm |
| RUY329 | MATaclb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm cdh1∷LEU2 |
| RUY332 | MATaclb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm orc6∷HIS3∷LEU2∷ORC6-ps,rxl |
| RUY334 | MATaclb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm orc6∷HIS3∷LEU2∷ORC6-ps,rxl cdh1∷LEU2 |
| RUY415 | MATaMCM7-NLS clb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm CLB6Δ3p-HA∷URA3 |
| RUY435 | MATarad17∷KanMX clb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm |
| RUY436 | MATarad17∷KanMX orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm |
| RUY437 | MATα rad17∷KanMX clb5∷URA3 orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm |
| RUY438 | MAT? mre11∷KanMX clb5∷URA3 URA3∷GAL-CDC6ΔNT-HAm |
| RUY439 | MAT? mre11∷KanMX orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm |
| RUY440 | MAT? mre11∷KanMX clb5∷URA3 orc6∷HIS3∷LEU2∷ORC6-ps,rxl URA3∷GAL-CDC6ΔNT-HAm |
| RUY299 | MATα orc6∷HIS3∷LEU2∷ORC6-ps,rxl MCM7-NLS URA3∷GAL-CDC6ΔNT-HAm rad17∷KanMX |
| RUY302 | MAT? orc6∷HIS3∷LEU2∷ORC6-ps,rxl MCM7-NLS URA3∷GAL-CDC6ΔNT-HAm mre11∷KanMX |
| RUY303 | MATaorc6∷HIS3∷LEU2∷ORC6-ps,rxl MCM7-NLS URA3∷GAL-CDC6ΔNT-HAm mre11∷KanMX rad17∷KanMX |
| RUY280 | MATaclb1 clb2(ts) clb3∷TRP1 clb4∷his3∷KanMX URA3∷GAL-CDC6ΔNT-HAm orc6∷HIS3∷LEU2∷ORC6-ps,rxl |
| Alleles used in this study | Description and references |
| ORC6-rxl | Linealized pRS405 (LEU marked)-based ORC6-rxl plasmid was integrated into orc6∷HIS3MX strain. ORC6-rxl contains mutations in RXL motif, which interfere with Clb5 binding (Wilmes et al. 2004). |
| GAL-CDC6ΔNT-HAs | Deletion of N-terminal region of CDC6 allows its stable expression due to insufficient phosphorylation by CDKs. The plasmid was kindly provided by J. Diffley. The single integration of GAL-CDC6Δ2-48-HA was confirmed by Southern blotting. Endogenous copy of CDC6 exists in addition to GAL-CDC6Δ2-48 in the strain (Archambault et al. 2005a,b). |
| ORC6-ps,rxl | ORC6-ps,rxl contains ORC6 Cdk phosphorylation site mutations in addition to ORC6-rxl. pRS405-based ORC6-ps,rxl plasmid was linealized and integrated into the orc6∷HIS3MX strain (Wilmes et al. 2004). |
| GAL-CDC6ΔNT-HAm | Multiple copy of GAL-CDC6Δ2-48-HA was integrated on the basis of Southern blotting. Otherwise the feature of this strain is as the same as that of GAL-CDC6ΔNT-HAs (Wilmes et al. 2004). |
| MCM7-NLS | Endogenous copy of Mcm7 was fused at its C terminus to two tandem copies of the SV40 nuclear localization signal allowing constitutive nuclear localization of Mcm7 (Nguyen et al. 2001). |
| CLB6Δ3P | Three serines or threonines of the phosphorylation sites in Clb6 were mutated to alanine, and the resulting CLB6Δ3P was expressed from the endogenous CLB6 promoter (Jackson et al. 2006). The CLB6Δ3P stabilizes Clb6p and escapes from SCF-dependent degradation. |