MBIO.4.13.6

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13 THE REPLICON (Full Edition)

6 Replication origins can be isolated in yeast

ARS (autonomous replication sequence) is an origin for replication in yeast. The common feature among different ARS sequences is a conserved 11 bp sequence called the A-domain.
The A domain is the conserved 11 bp sequence of A-T base pairs in the yeast ARS element that comprises the replication origin.

Origins in S. cerevisiae are short A•T-rich sequences that have an essential 11 bp sequence.
The ORC is a complex of 6 proteins that binds to an ARS.

Any segment of DNA that has an origin should be able to replicate. So although plasmids are rare in eukaryotes, it may be possible to construct them by suitable manipulation in vitro. This has been accomplished in yeast, although not in higher eukaryotes.

S. cerevisiae mutants can be "transformed" to the wild 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 ARS (for autonomously replicating sequence). ARS elements are derived from origins of replication.

Where ARS elements have been systematically mapped over extended chromosomal regions, it seems that only some of them are actually used to initiate replication. 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.

Figure 13.10

An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1-B3).

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 may be important in the rest of the region. Figure 13.10 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, that 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 (541).

>Mutations in three adjacent elements, numbered B1-B3, reduce origin function. An origin can function effectively with any 2 of the B elements, so 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 ORC (origin recognition complex) is a complex of 6 proteins with a mass of ~400 kD (for review see 2222). ORC binds to the 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 Licensing factor consists of MCM proteins). 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 (2247). This means that the average length of a replicon is ~35,000 bp. Counterparts to ORC are found in higher eukaryotic cells (2199).

ORC was first found in S. cerevisiae (where it is called scORC), but similar complexes have now been characterized in S. pombe (spORC), Drosophila (DmORC) and Xenopus (XlORC). All of the ORC complexes bind to DNA. Although none of the binding sites have been characterized 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 (for review see 3089). However, details of the interaction are clear only in S. cerevisiae; it is possible that additional components are required to recognize the origin in the other cases.

ARS elements satisfy the classic definition of an origin as a cis-acting sequence that causes DNA replication to initiate. Are similar elements to be found in higher eukaryotes? 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 conservation of sequence among putative origins in different organisms (for review see 4186).

Difficulties in finding consensus origin sequences cells suggest the possibility that origins may be more complex (or determined by features other than discrete cis-acting sequences). There are suggestions that some animal cell replicons may 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 (for review see 116).

A reconciliation between this phenomenon and the use of ORCs is suggested by the discovery that environmental effects can influence the use of origins (4185). At one location where multiple bubbles are found, there is a primary origin that is used predominantly when the nucleotide supply is high. But when the nucleotide supply is limiting, 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 higher eukaryotes.

Last Revised on October 19, 2004

116 DePamphlis, M. L. (1993). Eukaryotic DNA replication: anatomy of an origin. Annu. Rev. Biochem. 62, 29-63. PubMed Journal
2222 Kelly, T. J. and Brown, G. W. (2000). Regulation of chromosome replication. Annu. Rev. Biochem. 69, 829-880. PubMed Journal
3089 Bell, S. P. and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333-374. PubMed Journal
4186 Gilbert, D. M. (2001). Making sense of eukaryotic DNA replication origins. Science 294, 96-100. PubMed

541 Marahrens, Y. and Stillman, B. (1992). A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255, 817-823. PubMed
2199 Chesnokov, I., Remus, D., and Botchan, M. (2001). Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proc. Natl. Acad. Sci. USA 98, 11997-12002. PubMed Journal
2247 Wyrick, J. J., Aparicio, J. G., Chen, T., Barnett, J. D., Jennings, E. G., Young, R. A., Bell, S. P., and Aparicio, O. M. (2001). Genome-Wide Distribution of ORC and MCM Proteins in S. cerevisiae: High-Resolution Mapping of Replication Origins. Science 294, 2357-2360. PubMed Journal
4185 Anglana, M., Apiou, F., Bensimon, A., and Debatisse, M. (2003). Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing. Cell 114, 385-394. PubMed

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