Creation of ARS activity in yeast through iteration of non-functional sequences.

Replication origins in Saccharomyces cerevisiae have been identified through the cloning of autonomous replication sequence (ARS) elements that allow the extrachromosomal maintenance of plasmid molecules. ARS activity requires a close match to an 11 bp consensus sequence and A + T-rich flanking DNA. ARS elements with a wide range of capacities for promoting plasmid maintenance have been described. We determined the ARS activity of plasmids with inserts consisting of repetitions of a 64 bp 100% A + T sequence that has sequence similarities to known ARS elements. An insert with approximately four repeats did not yield transformants, but inserts with either eight or eleven repeats did. The cooperative production of ARS activity did not require a contiguous arrangement since a plasmid containing two inserts of four repeats each, separated by about 1 kb, was functional. Our results show that a change from non-function to function can be accomplished by the cumulative action of individually inactive sequences. We conclude that the probability of replication initiation is too low with only four repeats to allow plasmid maintenance, but the overall probability is increased by further sequence iteration to provide origin activity. We suggest that chromosomes may contain stretches with dispersed, weak origin elements, each undetected by the conventional ARS assay, that in sum provide origin function.