Academic journal article Genetics

The Temporal Program of Chromosome Replication: Genomewide Replication in clb5[Delta] Saccharomyces Cerevisiae

Academic journal article Genetics

The Temporal Program of Chromosome Replication: Genomewide Replication in clb5[Delta] Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Δ strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.

DNA replication in eukaryotic cells is a complex enterprise that appears to be optimized for anything but speed. Although each chromosome has multiple origins that are capable of supporting replication initiation, the number of origins that actually initiate DNA synthesis ("fire") varies. Some origins are efficient, firing in virtually every cell in a population, while others are inefficient, firing in only a subpopulation of cells. Thus, the potential density of active origins is greater than the actual density. Furthermore, those origins that fire appear not to do so simultaneously. A few, well-characterized origins have been shown to fire later in S phase (Ferguson et al. 1991; Friedman et al. 1997; Yamashita et al. 1997). Therefore, the density of origins, their efficiencies of activation, the times at which they fire, and the relative rates at which forks move should determine how long it takes to fully replicate a eukaryotic genome and the order in which it is replicated.

Recent analysis of Saccharomyces cerevisiae chromosome VI replication by in vivo labeling and singlemolecule analysis by DNA combing (Czajkowsky et al. 2008) suggested that there is no obligate order of origin firing along any single chromosomalDNAmolecule and that the observed temporal pattern of replication for a population of molecules could be explained largely by variable probabilities of origin firing without the need to invoke temporal staggering of initiations at different origins. A similar suggestion has been made regarding origin firing in fission yeast and metazoans (Rhind 2006). DNA fiber images are compelling, but their analysis is not entirely straightforward, requiring large sample sizes and different pulse/chase regimens to capture the complexity of the population. Nevertheless, they do raise questions about the conclusions of previous work, which all pointed to the existence of a temporal firing pattern. In fact, the results of some previous studies could be interpreted either way-as a temporal firing pattern or as population aggregates of origins with different efficiencies. For example, mapping of single-stranded DNA (ssDNA) after hydroxyurea treatment identified origins that are either unchecked or checked by the checkpoint protein Rad53p (Feng et al. 2006) largely along the lines of what previously had been classified as early- vs. late-replicating origins, respectively. However, the relative abundance of ssDNA at different origins matched more closely with origin efficiency than with replication time for the few origins for which efficiency had been measured.

We started by considering the two extreme possibilities: (1) that, as proposed for Schizosaccharomyces pombe (Patel et al. …

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