Proc Natl Acad Sci U S A 102(2): 337-342

DNA replication origins in the <em>Schizosaccharomyces pombe</em> genome

^{Department of Molecular Biology and Genetics, The Johns Hopkins University, Baltimore, MD 21205; and}^{Laboratory of Regulation of DNA Replication, Program in Molecular Biology, Sloan–Kettering Institute, New York, NY 10021}

^{To whom correspondence should be sent at the ‡ address. E-mail:}gro.ccksm@yllekt.

^{Present address: J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850.}

Contributed by Thomas J. Kelly, November 26, 2004

Freely available online through the PNAS open access option.

Abstract

Origins of DNA replication in Schizosaccharomyces pombe lack a specific consensus sequence analogous to the Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, raising the question of how they are recognized by the replication machinery. Because all well characterized S. pombe origins are located in intergenic regions, we analyzed the sequence properties and biological activity of such regions. The AT content of intergenes is very high (≈70%), and runs of A's or T's occur with a significantly greater frequency than expected. Additionally, the two DNA strands in intergenes display compositional asymmetry that strongly correlates with the direction of transcription of flanking genes. Importantly, the sequence properties of known S. pombe origins of DNA replication are similar to those of intergenes in general. In functional studies, we assayed the in vivo origin activity of 26 intergenes in a 68-kb region of S. pombe chromosome 2. We also assayed the origin activity of sets of randomly chosen intergenes with the same length or AT content. Our data demonstrate that at least half of intergenes have potential origin activity and that the relative ability of an intergene to function as an origin is governed primarily by AT content and length. We propose a stochastic model for initiation of DNA replication in the fission yeast. In this model, the number of AT tracts in a given sequence is the major determinant of its probability of binding SpORC and serving as a replication origin. A similar model may explain some features of origins of DNA replication in metazoans.

Abstract

The replicon model postulates that initiation of DNA replication takes place at specific chromosomal sequence elements (replicators) that are recognized by regulatory proteins (initiators) (1). This model was originally proposed to explain features of the replication of prokaryotic cells and viruses and has been validated in such systems by a large body of evidence (2). In eukaryotic cells, DNA replication is initiated from hundreds to thousands of different chromosomal sites in each cell cycle, raising the question of whether the replicon model provides a valid description of the initiation process. Although early genetic studies indicated that DNA replication in the budding yeast Saccharomyces cerevisiae conforms to the main features of the model, it is not yet clear that this is the case for most other eukaryotic species.

Origins of DNA replication in S. cerevisiae were identified as sequence elements that conferred the property of autonomous replication on extrachromosomal plasmids (3). Genetic analysis demonstrated that such autonomously replicating sequences (ARS) were ≈100 bp in length and contained a common 11-bp consensus sequence essential for origin activity, as well as other sequences that augmented origin activity (3–7). The characterization of S. cerevisiae ARS elements led to the identification of the yeast origin recognition complex (ScORC), the initiator protein that recognizes the ARS-consensus sequence (8). The specificity of the interaction of ScORC with origins is quite high. Single base substitutions are sufficient to abolish ScORC binding to the consensus sequence and prevent initiation of DNA replication (8, 9).

Origins of DNA replication in Schizosaccharomyces pombe differ in a number of ways from those of S. cerevisiae (10–17). They are very large (>500 bp) and extremely AT-rich. Although they often contain asymmetrically distributed clusters of A's and T's, they do not contain a highly specific consensus sequence analogous to the S. cerevisiae ARS consensus. Genetic studies have shown that S. pombe origins contain multiple redundant elements, which can be deleted or replaced by other AT-rich sequences without significantly affecting activity (14–16, 18). Most of the chromosomal origins that have been analyzed in S. pombe fire in only a minority of cell cycles, suggesting that the number of potential origins is greater than the number actually used in any given cell cycle (11, 13, 17, 19–21). These properties are not easily reconciled with the classical replicon model.

The sequencing of the S. pombe genome has made it possible to begin studying the distribution of chromosomal origins of DNA replication (22). Because almost all known S. pombe origins are located in intergenes, we studied the sequence properties and biological activity of such regions. Bioinformatic analysis revealed that the sequences of intergenes (and introns) are not random but exhibit certain underlying patterns that presumably reflect the nature of the mutational mechanisms that operate on the S. pombe genome. The sequence properties of origins of DNA replication are similar to those of intergenes in general. Functional studies of a 68-kb region of S. pombe chromosome 2 demonstrated that more than half of intergenes have potential origin activity and that the relative ability of an intergene to serve as an origin is a function of both its AT content and its length. Based on these and other data we propose that initiation of DNA replication in S. pombe (and perhaps metazoans) conforms to a stochastic model rather than the classical replicon model.

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Acknowledgments

We thank Pamela Simancek and Deborah Tien for technical assistance and the other members of the Kelly laboratory for stimulating discussions. This work was supported by National Institutes of Health Grants CA40414 and GM50806.

Acknowledgments

Notes

Author contributions: J.D., R.-Y.C., and T.J.K. designed research; J.D. and T.J.K. performed research; J.D., R.-Y.C., and T.J.K. contributed new reagents/analytic tools; J.D. and T.J.K. analyzed data; and J.D. and T.J.K. wrote the paper.

Abbreviations: ARS, autonomously replicating sequence; ORC, origin recognition complex.

Notes

Abbreviations: ARS, autonomously replicating sequence; ORC, origin recognition complex.

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