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. INTRODUCTIONEukaryotic DNA replication begins at discrete origins distributed along chromosomes. Although much progress has been made in understanding the mechanisms that establish and activate individual origins, the manner in which origin firing is coordinately regulated spatially along the chromosomes and temporally throughout S phase remains unclear (Kelly and Brown, 2000;Gilbert, 2001;Schwob, 2004). These questions are of particular interest because in most cases origin firing is not efficient; that is, a particular origin will fire in only a fraction of cell cycles. The observation that origins do not fire in every cell cycle raises the question of how individual origins are selected to fire and if that selection is distributed in a coordinated manner. In the absence of such coordination, origin firing would be randomly distributed, leading to the random gap problem; some cells would have large gaps between origin firing that would take a long time to replicate (Lucas et al., 2000;Herrick et al., 2002;Hyrien et al., 2003;Jun et al., 2004). Simple models of replication kinetics predict that if replication origins are randomly distributed, ϳ5% of the cells will have such large gaps between active origins that they will take four times longer than average to replicate (see Materials and Methods for calculations). Alternatively, cells could have a mechanism that evenly distributes origin firing across the genome; they would thus avoid the random gap problem, and replication would be efficient. Models for such mechanisms have been proposed; for instance, origins within specific clusters could be selected to fire, or active origins could suppress their neighbors by lateral inhibition (Mesner et al., 2003;Shechter and Gautier, 2005). However, little direct evidence exists to either support or refute these models.Origin structure and function has been well characterized in the budding yeast Saccharomyces cerevisiae (Kelly and Brown, 2000;Gilbert, 2001). Budding yeast have small (ϳ100 base pairs) origins characterized by a 17-b...

Genome-wide characterization of fission yeast DNA replication origins

“…In the 'time-course experiment', DNA samples were taken every 5-10 min until S-phase was complete. In the 'HU experiment', HU was added to slow down replication fork progression and confine DNA synthesis to the vicinity of origins (Patel et al, 2006). DNA samples were taken from cells that had been blocked for 90 min in HU, which is around 30 min after the onset of S-phase when bulk DNA replication was almost completed in a cdc25-22 block/release experiment ( Figure 1A, right panel).…”

“…An example of the timecourse experiment for a 300 kb region is given in Figure 2A. The HU experiment allowed origins to be mapped more precisely because fork migration away from the origin was reduced (Patel et al, 2006). A moving average across three microarray probes was applied (B3.9 kb) and signal ratios were plotted along the chromosomes.…”

DNA replication timing: random thoughts about origin firing

“…To avoid the random gap problem, it has been argued that there must be some mechanism to coordinate origin firing so as to avoid large random gaps 22 . Surprisingly, in two cases in which the distribution of origin firing has been carefully examined -frog embryo extracts and fission yeast -the distribution was in fact random and large random gaps were observed 8,9,23,24 .…”

“…The fact that budding yeast more closely fits the replicon model has made it much easier to understand replication in budding yeast, and has supported application of the replicon paradigm to eukaryotes in general. However, the observation that budding yeast's strategy of well-spaced, efficient origins is not conserved in its distant cousin, fission yeast, let alone in metazoans, suggests that it does not serve as a general model for the global regulation of eukaryotic origins 8,13,14 . Thus, budding yeast seems to be the exception, rather than the rule, in the organization of eukaryotic replication.…”

Spatio-Temporal Organization of Replication in Bacteria and Eukaryotes (Nucleoids and Nuclei)

“…But even in this system, analysis of DNA from single cells shows that initiation can be intrinsically disordered in time (Czajkowsky et al 2008), implying that origin selection has a significant stochastic component (Patel et al 2006). In mammalian cells, origin selection is also known to be highly stochastic (see Maya-Mendoza et al 2009 for review).…”