Genome-wide characterization of fission yeast DNA replication origins - PubMed (original) (raw)
Comparative Study
. 2006 Nov 1;25(21):5171-9.
doi: 10.1038/sj.emboj.7601390. Epub 2006 Oct 19.
Affiliations
- PMID: 17053780
- PMCID: PMC1630417
- DOI: 10.1038/sj.emboj.7601390
Comparative Study
Genome-wide characterization of fission yeast DNA replication origins
Christian Heichinger et al. EMBO J. 2006.
Abstract
Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.
Figures
Figure 1
Assessment of S-phase kinetics for microarray experiments. (A) Left panel: FACS analysis of a synchronous mitotic S-phase. Cells were synchronized by incubating the temperature-sensitive cdc25-22 mutant strain at the restrictive temperature, which blocked cells in late G2. On shift to the permissive temperature, these cells rapidly underwent mitosis and G1, and entered S-phase synchronously. It should be pointed out that in fission yeast cytokinesis takes place in late S-phase/early G2. Therefore, the DNA content when cells enter S-phase as seen by FACS analysis is 2C (binucleate cells) and increases to 4C before cells divide. Right panel: Replication kinetics of a synchronous cell population in mitotic S-phase (see Materials and methods). Bulk DNA replication takes place between 60 and 100 min. (B) Comparison of HU replication profiles of a 90 and 120 min time point. A 500 kb region of Chromosome 2 is shown. The signal ratios do not significantly change when cells are blocked for an additional 30 min in HU.
Figure 2
Mapping origins in the mitotic S-phase of fission yeast. (A) Replication timing profile of the time-course experiment along a 300 kb region of Chromosome 3. A moving average of the time at which 50% of each probe was replicated was determined (see Materials and methods) and plotted against chromosome position. (B) Replication profile of a triplicate repeat of the HU experiment; same region as in (A) is shown. Averaged microarray signal ratios were plotted for each probe against chromosome position. The plot of a self/self-hybridization is shown as a control. (C) Overlay of (A) and (B). Arrows indicate the position of origins in this region.
Figure 3
Origin firing in a _rad3_Δ mutant. Comparison of wild type and _rad3_Δ replication profile from HU experiments of a 300 kb region from Chromosome 3. Cells were blocked in HU for 90 min after release from G2 (see Materials and methods).
Figure 4
Comparison of interorigin distances in fission yeast and budding yeast. Histogram of the interorigin distance for 401 origins, the 904 peaks identified in the HU experiment and 332 origins of budding yeast (Raghuraman et al, 2001).
Figure 5
Mapping of origins in premeiotic S-phase. (A) Left panel: FACS analysis of a synchronous premeiotic S-phase (see Materials and methods). Right panel: Replication kinetics of a synchronous cell population in premeiotic S-phase (see Materials and methods). Bulk DNA replication takes place between 90 and 150 min. (B) Comparison of replication profiles from the time course and HU experiments in premeiotic S-phase. A 300 kb region of Chromosome 3 is shown. (C) Mitotic and meiotic replication profiles of the HU experiment are superimposed along the region shown in (B). Arrows indicate positions of origins.
Figure 6
Comparison of origin efficiencies from single molecule and microarray analyses. Origin efficiencies estimated from 15 origins analyzed on single DNA molecules (Patel et al, 2006) were plotted against origin efficiency estimates from our microarrays.
Figure 7
Origin efficiencies in mitotic and premeiotic S-phase. (A) Origin efficiency is higher in mitotic than in premeiotic S-phase. The signal ratio was used to estimate the efficiency of 401 origins in meiosis and mitosis. A histogram of efficiency distribution is shown. (B) Origin efficiency and time of replication shows a negative correlation in mitotic S-phase. The efficiency of origins was plotted as a function of origin replication time. (C) Two large domains (∼1.3 Mb) of efficient mitotic origins on Chromosome 1 are marked with brackets. (D) Clusters of origins (black bars) that are at least two-fold induced in premeiotic S-phase are indicated (brackets). Origins that are more than 35% efficient in mitotic S-phase were plotted on the same line below (gray bars). The position of centromeres is marked with a dot.
Figure 8
Replication fork velocities in mitotic and premeiotic S-phase. Replication fork velocity was determined from the replication timing profiles by measuring the gradient of replication forks from 31 mitotic and 38 meiotic origins.
Figure 9
Characterization of a large interorigin region. (A) Overlay of the HU and time-course experiments across a 300 kb region of Chromosome 2. Origins flanking the 140 kb region analyzed (as indicated) are marked by two large arrows. Small arrows indicate distinct but small peaks (signal ratios below 1.1) within the 140 kb region on the replication profiles of the HU experiment. (B) Replication profiles of a triplicate repeat of the HU experiment of the 300 kb region shown in (A). Even small peaks within the 140 kb region were reproducible.
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