Replication enhancers in fruit flies control the programmed amplification (extra rounds of replication in non-cycling cells) of chorion gene clusters on the third and X chromosomes. On the third chromosome, several cis-acting sequences, ACE3 and AER-a through AER-d, are essential for amplification [1, 2]. Of these, ACE3 is quantitatively the most important. Its ability to stimulate amplification is both orientation- and (within 1.5 kbp) distance-independent. It has been localized to a 320-bp region, and the effects of internal deletions suggest that it contains multiple partially redundant internal domains [2]. Whether it can stimulate non-chorion replication origins has not, to our knowledge, been tested.

In budding yeast, replication enhancers have been found near several different chromosomal replication origins. In several cases, the enhancers bind transcription factors [3,4,5]. In all cases tested so far, budding yeast enhancers have proved to be origin-specific; they stimulate only the origin with which they are normally associated in the chromosome [5, 6].

The replication enhancer that has been studied in fission yeast is part of the ura4 origin cluster - a group of three origins, called ars3003, ars3002 and ars3004 in their order from left to right upstream of the ura4 gene in the left portion of chromosome III (green boxes in Fig. ​Fig.1;1; [7,8,9]). We recently found that ars3002 and ars3004 overlap by ~400 bp (between the EcoRI and HincII sites, Fig. ​Fig.1;1; [7]). The overlap region is essential for ars3004 to function as a replication origin in plasmids. In contrast, the ~800-bp EcoRV-EcoRI fragment containing core ars3002 (without the overlap) is sufficient for plasmid origin function. However, addition of the overlap region to core ars3002 dramatically increases its plasmid origin activity [7]. Moreover, although the overlap region has no plasmid origin activity of its own, it strongly stimulated the plasmid origin activity of all other tested origins (ars3001 and ars3003), and it did so in a relatively distance- and orientation-independent fashion [7]. For these reasons, we call the overlap region a "replication enhancer" (yellow RE in Fig. ​Fig.1).1). We consider the enhancer to be a functional part of both ars3002 and ars3004. We found that the enhancer could not be substituted by a DNA fragment of the same size and base composition but different nucleotide sequence [7]. This observation suggests that specific nucleotide sequences within the enhancer are required for its function.

We have previously shown that ars3002 is the most active of the three replication origins in the ura4 origin cluster on the left end of fission yeast chromosome III (Fig. ​(Fig.1;1; [7, 8]). We have also shown that core ars3002 is no more active as a plasmid replication origin than is ars3003, whereas extended ars3002 is more active than ars3003 in the chromosome because the core activity is stimulated by the adjacent replication enhancer (Fig. ​(Fig.1),1), which is also part of ars3004 but which has no plasmid replication origin activity of its own [7].

As a first step toward understanding the mechanism of replication enhancement, we have generated consecutive ~50-bp deletions throughout the ura4 replication enhancer (Fig. ​(Fig.2),2), and we have tested the effects of these deletions on the activity of ars3002 as a plasmid replication origin (Fig. ​(Fig.3a).3a). We also tested the effects of ~50-bp deletions within core ars3002. The effects of these deletions within core ars3002 had previously been measured for core ars3002 by itself (Fig. ​(Fig.3b;3b; [10]). Here we determined their effects for extended ars3002 (core ars3002 plus enhancer). These experiments showed that enhancers deleted for each of the ~50-bp stretches within the enhancer were nevertheless capable of stimulating core ars3002. Thus, none of the ~50-bp stretches within the enhancer is essential for enhancer activity. However, deletion Δ16 at the core-proximal end of the enhancer significantly reduced plasmid origin activity as measured by the colony size assay (Fig. ​(Fig.3a3a).

These experiments also demonstrated that many of the ~50-bp stretches that are important for core ars3002 function (Fig. ​(Fig.3b)3b) are not important for extended ars3002 function. The ~50-bp stretch that proved most important for extended ars3002 function is region 10. Its deletion (Δ10 in Fig. ​Fig.3a)3a) dramatically reduced plasmid origin function by both the transformation frequency and colony size assays. Region 10 contains a stretch of 20 consecutive adenines in the top strand (Fig. ​(Fig.2).2). Short runs of adenines and thymines are also present in multiple locations elsewhere in ars3002 (Fig. ​(Fig.2).2). These may be binding sites for the S. pombe Origin Recognition Complex (ORC), whose fourth largest subunit (Orp4) contains a multiple AT-hook domain, which permits it to bind with high affinity to DNA containing runs of adenines or thymines in one strand [14, 15]. In fact, the largest subunit of S. pombe ORC (Orp1) has been mapped by chromatin immunoprecipitation to ars3002 and also to ars2004, which, like ars3002, contains multiple asymmetric A+T-rich stretches [16, 17].

We do not yet know the functions of the other sequences (regions 11-14 and 16-20) that-according to the results in Fig. ​Fig.3-also3-also contribute (though to a lesser extent than region 10) to the plasmid origin activity of extended ars3002. What is clear, however, is that the functions of all of the ~50-bp stretches are at least partially redundant. None of the deletions, even Δ10, eliminates origin activity. In each case, other sequences within extended ars3002 must be capable of providing at least a portion of the function of the deleted region. The redundant nature of the sequences within S. pombe replication origins has previously been amply noted [10, 11, 16, 18]. However, this is the first time that redundancy has also been found within a yeast replication enhancer. ACE3, the amplification enhancer for the chorion gene cluster on chromosome 3 of Drosophila melanogaster, also contains multiple redundant sequence elements [2].

In an attempt to reduce the redundancy in the enhancer and identify combinations of ~50-bp deletions that, together, would completely eliminate replication enhancer function, we constructed double and triple deletions so that each ~50-bp stretch was deleted in combination with one or two flanking ~50-bp stretches to the left and to the right. These analyses showed that the double deletions Δ16-17 and Δ20-21 were especially effective at reducing origin activity (Fig. ​(Fig.5a),5a), and the triple deletions Δ16-18 and Δ19-21 were even more effective (Fig. ​(Fig.5b).5b). In fact, these two triple deletions reduced the activity of extended ars3002 to the level of core ars3002. In other words, they abolished enhancer activity. The fact that the enhancer contains two non-overlapping essential ~150-bp regions (16-18 and 19-21) indicates that these two regions cooperate with each other to generate enhancer function. Neither provides complete enhancer function by itself. Each is essential for enhancer function. However, none of the three ~50-bp regions that together comprise each essential ~150-bp region is individually essential, suggesting internal redundancy within both of the ~150-bp essential regions.

What could be the sequences that contribute to enhancer function within these essential regions? Several laboratories, including ours, have identified consensus sequences that are often found in S. pombe replication origins [9,10,11, 13, 18]. All of these are A+T-rich and are asymmetric in the sense that most of the adenines are in one strand and most of the thymines are in the complementary strand. Previous origin deletion scans (similar to those in Fig. ​Fig.3)3) have shown that the regions most important for S. pombe origin function frequently contain clusters of these consensus sequences [10, 11, 16, 18]. To test whether the ars3002 replication enhancer is similarly organized, we scanned it for near matches to the four characterized consensus sequences [9,10,11, 13, 18]. We found several potentially interesting consensus matches within the important portions of the enhancer (Fig. ​(Fig.6).6). In region 16 (the ~50-bp region that seems most important for enhancer function; Fig. ​Fig.3a),3a), there is a 10/11 match to the consensus sequence identified by Maundrell et al. [13], which is highlighted with light green in Fig. ​Fig.6.6. Although this consensus, like the three others, has not been found to be completely essential for any tested S. pombe origin, a 10-bp linker substitution in ars3001 (the S. pombe rDNA origin) that removes a Maundrell consensus sequence reduces origin activity about 30-fold [11]. Consequently, there is a good chance that this sequence contributes significantly to whatever role region 16 plays in enhancer function.

Spanning the junction of regions 20 and 21 is a 20/30 match to a 30-bp sequence found by Clyne and Kelley [18] to be important for ars1 plasmid replication origin activity (blue in Fig. ​Fig.6).6). In addition, region 21 contains several overlapping 11/12 matches to the consensus sequence of Zhu et al. [9, 11], highlighted with light magenta in Fig. ​Fig.6.6. Overlapping matches to this consensus are also found in region 10 (Fig. ​(Fig.2),2), which is highly important for ars3002 function (Fig. ​(Fig.3;3; [9, 11]). These two consensus sequences are therefore good candidates for playing important roles in the enhancer activities mediated by regions 20 and 21.

Note that all three of the consensus sequences and also many additional regions within the enhancer contain runs of adenines or thymines and thus are potential binding sites for S. pombe ORC. It has recently been demonstrated that the Drosophila amplification enhancers, ACE3 and AER-d, bind Drosophila ORC [19]. Thus it is possible that ORC binding is an aspect of enhancer function conserved from fission yeast to animals.