Replication factories in budding yeast
A. Replication factories are found specifically during S phase. Homozygous POL1-4GFP (T3030), homozygous POL2-4GFP (T3031) and heterozygous YFP-PCNA/PCNA⁺ (T3060) diploid cells were cultured asynchronously. Top: representative images of bright-field and fluorescence, classified by the bud size. Scale bar: 2 μm. Bottom: percentages of cells with classified bud sizes, showing the fraction of fluorescence with a bright globular (red) or perinuclear (pale blue) pattern. n: numbers of observed cells with each classified bud size.
B. Formation of replication factories is dependent on B-type cyclin-CDKs. POL1-4GFP clb5Δ clb6Δ (T3262) homozygous diploid cells were cultured asynchronously and observed as in A.
C. Formation of replication factories is dependent on DNA replication. POL1-4GFP GAL-CDC6 cdc6Δ (T3264) homozygous diploid cells were grown in galactose plus raffinose-containing medium. After culture in raffinose-containing medium lacking galactose for 2.5 hrs, small unbudded cells were collected by elutriation, and incubated (start of incubation: 0 min) in either galactose/raffinose- (Cdc6+) or glucose- (Cdc6−) containing medium. Top: representative images of bright-field and fluorescence, classified by the bud size. Bottom: percentages of cells with buds (black line) and of small-budded cells with weak (white bar) or bright globular (red bar) Pol1 GFP signals. See .

Determining replication timing by time-lapse microscopy
A. Map of the integration sites of tet and lac operators. The replication timing profile of the chromosome region was taken from .
B-E. MATa P_GPD1-thymidine kinase (5 copies) P_ADH1-ENT1 tetR-3CFP GFP-lacI haploid cells with tetO×224 and lacO×256 (as indicated in A: T3765) were arrested by α factor treatment, washed, and released in BrdU-containing medium. After 20 min, samples were collected for time point 0 min, and other time point samples were subsequently collected. B. The budding index and the intensity of each tetOs-1 (red) and lacOs (green) dot of individual cells at different times are shown. Key: dimmer and brighter dots are schematically shown by small and large dots, respectively. The intensity of the dots was investigated in 128-300 cells at each time point. C. BrdU-labelled DNA was amplified by PCR using primers for the ARS (replication origin) and tetOs-1/lacOs integration sites. Throughout the time course, DNA template derived from the same number of cells was used for PCR amplification. If no BrdU was added to the culture there was no increase observed in the PCR products while the budding index increased (data not shown). If cells were treated with HU, there was an increase observed in PCR products for the ARS, but not for the tetOs-1 or lacOs site, while the budding index increased (data not shown). D. Quantification of the intensity of the bands in C. The intensity at time point 0 and the maximum intensity were set to 0 and 100 %, respectively. E. Cellular DNA content was measured by FACS.

DNA replication of chromosomal loci in replication factories.
A. Model of a closely associated double replisome at a replication factory, and expected behaviours of tetOs-1-CFP dots.
B-C. MATa POL1-4GFP tetR-3CFP haploid cells with tetO×224 (tetOs-1: as indicated in , but without the lacOs dot: T3639) were arrested by α factor treatment, washed, released into fresh medium and observed by time-lapse. Representative images (B, top) of Pol1-4GFP signals and tetOs-1-CFP dots, and fluorescence intensity of the dot during observation (B, bottom). Time is shown relative to replication (0 min: mid-point of the increase in the dot intensity on the regression curve). Inverted triangles indicate time points when colocalization between the tetOs-1-CFP dot and Pol1-4GFP bright globular signals (replication factories) was found. The percentage of cells (out of all observed cells) showing such colocalization at each time point (relative to replication: C). Scale bar: 1 μm. For individual data of all observed cells see .

Behaviour of two chromosomal loci on the opposite sides of a replicon with similar replication timing
A. Model of a closely associated double replisome at a replication factory, and expected behaviour of two chromosomal loci (tetOs-1 and lacOs: top), whose positions are shown below. The position of a control locus tetOs-2 is also indicated.
B. MATa tetR-3CFP GFP-lacI haploid cells with tetO×224 (tetOs-1) and lacO×256 (lacOs: as indicated in A: T3580) were arrested by α factor treatment, released into fresh medium and observed by time-lapse microscopy. CFP (red), GFP (green) and bright field images of a representative cell (top). Signal intensity of tetOs-1 (red) and lacOs (green) dots, and distance between the two dots (blue bars: bottom). Time is shown relative to mid-replication time (see text). Scale bar: 1 μm.
C. Replication time of tetOs-1 and lacOs dots was measured as in B and shown in closed red and green circles, respectively, for each cell (top). Time is shown relative to mid-replication (see text). Times of close localization of the two dots (≤ 350 nm, ≥ 2 min) are indicated by a blue line (top). The percentage of cells with close localization of the two dots is shown for each time window by a pink bar (bottom). Images of the cell #1 were shown in B. For individual data of all observed cells see .
D. MATa tetR-3CFP GFP-lacI haploid cells with tetO×224 (tetOs-2) and lacO×256 (lacOs: as indicated in A: T3959) were treated as in B. Replication time of the two dots is shown in open red (tetOs-2) and closed green (lacOs) circles for each cell (top). Percentage of the cells showing close localization of the two dots is shown as in C (bottom). For individual data of all observed cells see .

Median distance between tetOs-1 and lacOs dots is reduced around mid-replication time.
A. Median distance (blue bar) between tetOs-1 and lacOs dots at each time point among 12 cells shown in Fig and . B. Median distance (blue bar) between tetOs-2 and lacOs dots at each time point among 9 cells shown in Fig and . The red lines show smoothed curves. Time is shown relative to mid-replication time.

Behaviour of two loci on the same or on homologous chromosomes in diploids.
A. MATa/a diploid A (T4275) and B (T4277) cells harboured tetO×224 (tetOs-1) and lacO×256 (lacOs) on homologous chromosomes and on the same chromosome, respectively, as indicated schematically. Both strains also had one copy of tetR-3CFP and GFP-lacI.
B. The MATa/a diploid A and B cells were arrested by α factor treatment, released to a fresh medium and observed by time-lapse microscopy, as in . Time of replication at tetOs-1 (red circles) and lacOs (green circles) and times of close localization (≤ 350 nm, ≥ 2 min) between the two dots (blue lines) are shown for each diploid A and B cell. For individual data of all observed cells see .