DNA replication origins fire stochastically in fission yeast

Abstract

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

Figure 1.

Origin firing is randomly distributed across the genome. (A) Representative combed DNA molecules. Nascent replication bubbles are labeled green with anti-BrdU. The DNA molecule is labeled red with anti-guanosine. The positions of the nascent bubbles are indicated below the image with a green line. Signals are scored as real incorporation patches if they are longer than 3 kb; smaller signals are assumed to be background. The top molecule is from the HU-block data set; the bottom is from the early-pulse data set. (B-J) Histograms of the combing data. (C, F, and I) The distribution of bubble lengths. The HU-blocked bubbles stall after 9.4 ± 5.7 kb, consistent with replication forks stalling after ∼5 kb. The early-pulse-labeled bubbles show a more heterogeneous distribution, averaging 19.7 ± 13.1 kb. The late-pulse labeled bubbles were selected to be <30 kb (see Materials and Methods). (D, G, and J) The distribution of interbubble distances. Best-fit exponential curves and associated R² values are shown. The dashed curves represent the best fit excluding the first data point. For all three histograms, the best Gaussian fit has R² < 0.6 (unpublished data).

Figure 2.

Origins in the ura4 region fire stochastically. (A) The ura4 region of chromosome III and representative molecules. The graphic represents the left end of chromosome III from nucleotides 29423-254496. Blue represents coding sequences, black noncoding sequences, green noncoding sequences predicted to be origins by AT richness (Segurado et al., 2003) and yellow ura4. Predicted origins are labeled above with the systematic nomenclature of Segurado et al. (2003). Three additional loci, labeled 3002.2, 3005.3, and 3005.5 and shown in pink, are just below the AT-threshold used for origin prediction. Origin pairs 3004/3005 and 3008/3009 are too close together to be reliably distinguished by combing, and each pair is treated as one origin. The red bars represent the FISH probes used to identify molecules from this region. Three representative molecules labeled with BrdU and identified with the FISH probes are shown. The BrdU signals scored as replication bubbles are indicated by green lines below the images. The length and continuity of all molecules was determined with anti-guanosine antibodies, which label DNA (unpublished data). (B) A graphic table of origin use in the ura4 region. Each row represents a DNA molecule; each column represents an origin in the region. A green box indicates the origin fired on that molecule; a black box indicates it did not; no box indicates that the fiber did not extend to include that origin. Only the 82% of replication bubbles that overlapped predicted origins are included. Below the table is the quantitation of the origin efficiencies, and the efficiencies when either the right or left neighbor fired. The high efficiency of 3004/5 is due to the fact that it actually represents three origins: 3004 (also known as ars3003) and two closely spaced origins in 3005 (ars3002 and ars3004; Dubey et al., 1994). The 71% efficiency of the three is consistent with an efficiency of ∼35% for each individual origin, consistent with 2D-gel analyses of the origins (Dubey et al., 1994). (C) A graphic table of origin use in the nmt1 region.

Figure 3.

The ability of an origin to fire is not epigenetically inherited. (A) The experimental strategy, as described in the text. (B) Representative molecules. The upper, red images are visualized with anti-CldU antibodies; the gaps in the CldU signal, indicating origins that fired only in the first cell cycle, are marked below by white rectangles. The lower, green images are the same molecules visualized with anti-IdU antibodies; the IdU-labeled patches, indicating origins that fired in the second cell cycle, are marked above by green rectangles.