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Although cellular origins of DNA replication are poorly understood in most eukaryotes, short chromosomal sequences have been cloned in the yeast Saccharomyces cerevisiae that enable plasmids to replicate along with the cellular chromosomes in the S phase of each cell cycle (1). Such autonomously replicating sequences (ARS's) have been shown by two-dimensional agarose gel electrophoresis techniques to be authentic origins of replication in plasmids and in cellular chromosomes (2, 3). Interestingly, only a subset of S. cerevisiae ARS's are active origins at their native chromosomal positions (3).
All ARS's of both classes contain a match to a degenerate "ARS consensus sequence," 5' (Data omitted) that is essential for ARS function (4-7). The sequence requirements of this element have been thoroughly characterized, and any one of numerous single point mutations in this sequence eliminates ARS function (6). Although the conservation of this element suggests that it is recognized by a sequence-specific DNA binding protein, no protein has yet been shown to function at this sequence. A single strand DNA binding protein that binds the T-rich strand of this sequence has been identified and purified (8), but is not known whether this protein functions in DNA replication.
A sequence (or sequences) flanking the consensus match is also essential for origin function. Such sequences have been poorly defined because deletion mutagenesis has often produced conflicting results. For example, deletion mutagenesis of an ARS revealed a strong dependence on the nature of the adjacent sequence (9). One effect of adjacent sequences that has been demonstrated is the suppression of ARS function when nucleosomes intrude on the origin (10). Despite the poor sequence definition, there have been several proposals for a role of the essential flanking region. Deletions that reduce origin efficiency have variously been correlated with the disruption of a DNA bend, a DNA unwinding element, weak homology to the ARS consensus sequence, and a nuclear scaffold attachment site (11-15). The binding of the protein ABFI has also been suggested to be important for ARS function. Although ABFi only binds to a subset of origins, deletions and substitutions that disrupt these ABFI binding sites reduce the maintenance of plasmids (16-19).
For our study of the structure of a yeast origin of DNA replication, ARS1 was chosen because it is...