Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories.

Yeast genetics and molecular biology

Yeast strain background

(W303), methods for yeast culture and for cell cycle synchronization using α factor treatment or elutriation, and the TetR-GFP/ tet and GFP-lacI/ lac operator system were described previously ( Piatti et al., 1996 ; Straight et al., 1996 ; Michaelis et al., 1997 ; Tanaka et al., 1997 ; Amberg et al., 2005 ). Cells were cultured at 25°C in YP medium containing glucose unless otherwise stated. POL1 and POL2 were tagged with 4 tandem copies of GFP at their C-termini, and PCNA with YFP at its N-terminus. All tagging was done at the original gene loci by PCR methods as previously described ( Prein et al., 2000 ; Maekawa et al., 2003 ), and all tagged genes were expressed with their authentic promoters. POL1 , POL2 and PCNA are all essential for DNA replication and cell growth ( Sugino, 1995 ; Waga and Stillman, 1998 ). Haploid cells containing POL1-4GFP and POL2-4GFP grew normally, suggesting that these tagged genes were functional. YFP-PCNA severely retarded haploid cell growth, indicating that the tagged construct was not fully functional. Therefore, we used haploid or homozygous diploid cells containing POL1-4GFP or POL2-4GFP , and diploid cells containing heterozygous YFP-PCNA (i.e. PCNA on the other homologous chromosome is not tagged), all of which showed normal growth. To integrate tetO × 224 and lacO × 256 arrays into chromosomal loci ( tetOs-1 , tetOs-2 and lacOs ) as shown in Fig 2A and 4A , K. lactis URA3 was first inserted at non-coding regions between YDL089w and YDL088c , YDL020c and YDL019c , YDL055c and YDL054c , respectively, using a one-step PCR method ( Amberg et al., 2005 ). Two 650-700 bp DNA fragments flanking the insertion sites were amplified by PCR and cloned into the pUC18 plasmid (GenBank/EMBL accession number L09136 ). tetO × 224 and lacO × 256 arrays were inserted between the two DNA fragments. Using these constructs, K. lactis URA3 was replaced with tetO × 224 and lacO × 256 arrays by negative selection against URA3 on culture plates containing 5-fluoroorotic acid ( Amberg et al., 2005 ). Correct insertion of URA3 and subsequent replacement were confirmed by PCR amplification of relevant chromosomal regions. TetR-3CFP and GFP-lacI constructs were as described previously ( Straight et al., 1996 ; Bressan et al., 2004 ). MATa/a diploid cells were made by expressing the HO gene from the GAL1-10 promoter in MATa/α diploid cells ( Herskowitz and Jensen, 1991 ). To facilitate BrdU incorporation ( Vernis et al., 2003 ), 5 copies of the herpes simplex thymidine kinase gene were expressed from GPD1 promoters ( Dahmann et al., 1995 ), and the human equilibrative nucleoside transporter 1 ( ENT1 ) gene (its codon usage was optimized for yeast) was expressed from ADH1 promoter (K Shirahige et al , unpublished).

Microscopy and image analyses

The general procedures for time-lapse microscopy were described previously ( Tanaka et al., 2005 ). Time-lapse images were collected at indicated time intervals at 23°C (ambient temperature) unless otherwise stated. Using the Deltavision microscope (Applied Precision), we acquired 5-9 (0.7 μm apart) z-sections, unless otherwise stated, which were subsequently deconvoluted and projected to two-dimensional images using SoftWoRx (Applied Precision) and Volocity (Improvision) software. To distinguish GFP and CFP signals, the JP4 filter set (Chroma) was used. GFP and CFP signals were quantified using Volocity. Replication factories were judged to colocalize with tetOs dots when Pol1-4GFP bright globular signals overlapped with tetOs -CFP dots on the focal plane for tetOs -CFP dots for two minutes or longer. To measure the distance between tetOs -CFP and lacOs -GFP dots, the distance between their centres was quantified in three dimensional space. Other methods FACS DNA content analyses, BrdU incorporation and subsequent chromatin immunoprecipitation, indirect immunostaining of epitope tags and incorporated BrdU were as described previously ( Pichler et al., 1997 ; Dimitrova et al., 1999 ; Lengronne et al., 2001 ; Amberg et al., 2005 ). Find more methods in Supplementary Note 5 .

Other methods FACS DNA content analyses, BrdU incorporation and subsequent chromatin immunoprecipitation, indirect immunostaining of epitope tags and incorporated BrdU were as described previously ( Pichler et al., 1997 ; Dimitrova et al., 1999 ; Lengronne et al., 2001 ; Amberg et al., 2005 ). Find more methods in Supplementary Note 5 .

Supplementary Material Suppl inf