Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.
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This paper is dedicated to J. Herbert Taylor, a leader in the field of DNA replication, who passed away in December 1998. Among his many contributions, he demonstrated by autoradiography that chromosomal DNA replication is semiconservative. We thank C. Liang and B. Stillman for providing the SP1 yeast strain, and J. Petrini for the gift of the cdc9 mutant strain. We are grateful to E. A. Hendrickson, S. Lange, and M. North for critically reading the manuscript. A.-K. B. thanks B. R. Calvi for many insightful and inspiring discussions and critical comments on the manuscript. This work was supported by NIH grant GM 35929 to S. A. G.