Replication Origins Can Be Isolated in Yeast

KEY CONCEPTS
- Origins in Saccharomyces cerevisiae are short A-T sequences that have an essential 11-bp sequence.
- The origin recognition complex is a complex of six proteins that binds to an autonomously replicating sequence.
- Related origin recognition complexes are found in multicellular eukaryotes.
Any segment of DNA that has an origin should be able to replicate, so although plasmids are rare in eukaryotes, it might be possible to construct them by suitable manipulation in vitro. Researchers have accomplished this in yeast, but not in multicellular eukaryotes. S. cerevisiae mutants can be “transformed” to the wild-type phenotype by addition of DNA that carries a wild-type copy of the gene. The discovery of yeast origins resulted from the observation that some yeast DNA fragments (when circularized) are able to transform defective cells very efficiently. These fragments can survive in the cell in the unintegrated (autonomous) state; that is, as self-replicating plasmids.
A high-frequency transforming fragment possesses a sequence that confers the ability to replicate efficiently in yeast. This segment is called an autonomously replicating sequence (ARS). ARS elements are derived from origins of replication.
Although ARS elements have been systematically mapped over extended chromosomal regions, it seems that only some of them are actually used to initiate replication at any one time. The others are silent, or possibly used only occasionally. If it is true that some origins have varying probabilities of being used, it follows that there can be no fixed termini between replicons. In this case, a given region of a chromosome could be replicated from different origins in different cell cycles.
An ARS element consists of an A-T–rich region that contains discrete sites in which mutations affect origin function. Base composition rather than sequence might be important in the rest of the region. FIGURE 1 shows a systematic mutational analysis along the length of an origin. Origin function is abolished completely by mutations in a 14-bp “core” region, called the A domain, which contains an 11-bp consensus sequence consisting of A-T base pairs. This consensus sequence (sometimes called the ACS, for ARS consensus sequence) is the only homology between known ARS elements.

FIGURE 1. An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1–B3).
Mutations in three adjacent elements, numbered B1 to B3, reduce origin function. An origin can function effectively with any two of the B elements, as long as a functional A element is present. (Imperfect copies of the core consensus, typically conforming at 9/11 positions, are found close to, or overlapping with, each B element, but they do not appear to be necessary for origin function.)
The origin recognition complex (ORC) is a highly conserved complex found in all eukaryotes. It is composed of six proteins with a mass of approximately 400 kilodaltons (kD). ORC binds to the yeast A and B1 elements on the A-T-rich strand and is associated with ARS elements throughout the cell cycle. This means that initiation depends on changes in its condition rather than de novo association with an origin (see the section Licensing Factor Binds to ORC later in this chapter). By counting the number of sites to which ORC binds, we can estimate that there are about 400 origins of replication in the yeast genome. This means that the average length of a replicon is approximately 35,000 bp. Counterparts to ORC are found in cells of multicellular eukaryotes.
ORC was first found in S. cerevisiae (where it is sometimes called scORC), but similar complexes have now been characterized in Schizosaccharomyces pombe (spORC), Drosophila (DmORC), and Xenopus (XlORC). All of the ORC complexes bind to DNA.
Although researchers have not characterized any of the binding sites in the same detail as in S. cerevisiae, in several cases, they are at locations associated with the initiation of replication. It
seems clear that ORC is an initiation complex whose binding identifies an origin of replication. Details of the interaction, however, are clear only in S. cerevisiae; it is possible that additional components are required to recognize the origin in the other cases. The yeast ARS elements satisfy the classic definition of an origin as a cis-acting sequence that causes DNA replication to initiate.

The conservation of the ORC suggests that origins are likely to take the same sort of form in other eukaryotes, but in spite of this, there is little to no conservation of sequence among putative origins in different organisms. Difficulties in finding consensus origin sequences suggest the possibility that origins might be more complex (or determined by features other than discrete cis-acting sequences). There are suggestions that some animal cell replicons might have complex patterns of initiation: In some cases, many small replication bubbles are found in one region, posing the question of whether there are alternative or multiple starts to replication and whether there is a small discrete origin. Replication origins are often associated with promoters of genes.
Reconciliation between this phenomenon and the use of ORCs is suggested by the discovery that environmental effects can influence the use of origins. At one location where multiple bubbles are found, there is a primary origin that is used predominantly when the nucleotide supply is high. When the nucleotide supply is limiting, though, many secondary origins are also used, giving rise to a pattern of multiple bubbles. One possible molecular explanation is that ORCs dissociate from the primary origin and initiate elsewhere in the vicinity if the supply of nucleotides is insufficient for the initiation reaction to occur quickly. At all events, it now seems likely that we will be able in due course to characterize discrete sequences that function as origins of replication in multicellular eukaryotes.