To understand the molecular mechanisms of initiation and regulation of eukaryotic DNA replication, it is helpful to study the cis
-acting sequences (replicators) essential for origin function. Identification and characterization of replicators in the budding yeast, Saccharomyces cerevisiae
, were facilitated by the discovery that DNA sequences serving as replicators in chromosomes also serve as replicators in plasmids (reviewed in reference 30
). These origin sequences are called autonomously replicating sequence (ARS) elements, because they permit plasmids to replicate autonomously in yeast cells.
Plasmids bearing ARS elements and selectable markers can transform S. cerevisiae
cells at a high frequency (15
), permitting a plasmid transformation assay that has been used to characterize the sequence requirements for origin function. ARS elements have two essential components: a short A domain of about 20 bp, which contains a ≥9-of-11 match to the 11-bp ARS consensus sequence (ACS) (4
), and a broad (~100-bp) flanking region, called the B domain, 3′ to the consensus T-rich strand. The B domain consists of two or three additional important sequence motifs (16
). One of the important sequences in the B domain, called B1, cooperates with the A domain to form a binding site for the origin recognition complex (ORC), the putative initiator protein (2
). Other possible functions for the B domain include serving as a DNA unwinding element (28
), enhancing origin activity by serving as a binding site for transcription factors (23
), and interacting with a single-stranded DNA binding protein (24
Replication origins in most other eukaryotes are poorly understood, mainly due to lack of unambiguous techniques for characterizing them. However, in the fission yeast, Schizosaccharomyces pombe
, which is evolutionarily distant from S. cerevisiae
), chromosomal DNA sequences with properties similar to ARS elements of budding yeast have been identified (25
), and some of them have been shown to correspond to chromosomal replication origins (8
). Because S. pombe
is in some respects more similar to other eukaryotic organisms than is S. cerevisiae
(reviewed in reference 45
), it is possible that further study of ARS elements in S. pombe
will provide information useful in understanding replication origins in many other eukaryotic organisms.
ARS elements are AT rich, like those of S. cerevisiae
, but are generally bigger (500 to 1500 bp). Two S. pombe
ARS elements, ars1
) and ars3002
), have been studied in some detail. Deletion and linker substitution analyses indicate that these ARS elements contain at least one (for ars1
) or two (for ars3002
) essential modules and some additional important modules, and each of the essential modules contains critical sequence elements extending for 20 to 30 bp. The critical sequences are all AT rich and asymmetric, in the sense that A residues are clustered on one strand while T residues are clustered on the complementary strand.
To test whether these features are common to other S. pombe
ARS elements, we chose to study the ribosomal DNA (rDNA) ARS element, which we have previously mapped to the nontranscribed spacer in the rDNA repeats (35
). Because there are 100 to 150 copies of the rDNA repeat in the S. pombe
genome, the rDNA ARS element is by far the most abundant ARS element in the genome.
We have previously shown that a 2.3-kbp Bam
I restriction fragment within the rDNA repeat (see Fig. ) exhibits as much ARS activity as a restriction fragment containing the entire rDNA repeat and that the 2.3-kbp fragment contains all detectable rDNA initiation sites (see the gray box in Fig. and reference 35
). The ARS element within this 2.3-kbp fragment was designated ars3001
according to the four-digit S. pombe
ARS-naming convention (8
), because it was the first ARS element to be discovered in chromosome III (9
FIG. 1 Summary of previous studies localizing ARS activity in the rDNA repeat. (Top) The rDNA repeat (10.9 kbp) consists of the 3.46-kbp nontranscribed spacer (NTS) and the transcribed region containing the external transcribed spacer (ETS); the 17S, 5.8S, and (more ...)
In this report, we describe the results of systematic mutagenesis of ars3001. These results identify three domains that are essential for function. Each of these domains contains important sequences which share similarities with those detected in the two earlier studies but are not equivalent to the ACS of S. cerevisiae ARS elements. Domain substitution experiments indicate that the three domains are largely orientation dependent and can partially substitute for each other.