Autonomously replicating sequence (ARS) elements were first identified in the budding yeast Saccharomyces cerevisiae as chromosomal DNA fragments that promoted high frequency of transformation and extrachromosomal maintenance of plasmid DNA. These specific sequence elements were subsequently shown to function as origins of DNA replication. Detailed analysis of the structure and function of ARS elements has been limited largely to S. cerevisiae and more recently the fission yeast Schizosaccharomyces pombe. Characterization of ARS activity in other eukaryotes is far less complete. Here we describe the ARS assay developed in yeast and its application to the study of origin function in other eukaryotes. Other available methods for detecting autonomous replication in these systems are also presented.

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.