This paper describes the first attempt in any organism to determine how the presence of a replication enhancer next to a core replication origin alters the relative importance of the sequences within the core origin. It also describes the first attempt in any yeast to map the nucleotide sequences important for function within a replication enhancer.
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. ; [7
]). 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. ), 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. ), and we have tested the effects of these deletions on the activity of ars3002
as a plasmid replication origin (Fig. ). 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. ; [10
]). Here we determined their effects for extended 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. ).
These experiments also demonstrated that many of the ~50-bp stretches that are important for core ars3002
function (Fig. ) 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. ) 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. ). Short runs of adenines and thymines are also present in multiple locations elsewhere in ars3002
(Fig. ). 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
]. 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
We do not yet know the functions of the other sequences (regions 11-14 and 16-20) that-according to the results in Fig. -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
]. 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. ), and the triple deletions Δ16-18 and Δ19-21 were even more effective (Fig. ). 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
]. 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. ) have shown that the regions most important for S. pombe
origin function frequently contain clusters of these consensus sequences [10
]. To test whether the ars3002
replication enhancer is similarly organized, we scanned it for near matches to the four characterized consensus sequences [9
]. We found several potentially interesting consensus matches within the important portions of the enhancer (Fig. ). In region 16 (the ~50-bp region that seems most important for enhancer function; Fig. ), there is a 10/11 match to the consensus sequence identified by Maundrell et al.
], which is highlighted with light green in Fig. . 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.
Figure 6 Nucleotide sequence of deletion regions 15-22 showing near matches to S. pombe replication origin consensus sequences. The sequence shown here is the same as in the bottom portion of Fig. . The light green 11-bp sequence is a 10/11 match (more ...)
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. ). In addition, region 21 contains several overlapping 11/12 matches to the consensus sequence of Zhu et al.
], highlighted with light magenta in Fig. . Overlapping matches to this consensus are also found in region 10 (Fig. ), which is highly important for ars3002
function (Fig. ; [9
]). 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
, bind Drosophila
]. Thus it is possible that ORC binding is an aspect of enhancer function conserved from fission yeast to animals.
Testing this hypothesis or any other hypothesis concerning the mechanism of enhancer function will require identifying and characterizing the proteins that bind to the cis
-acting sequences described here. During the past decade much progress has been made in identifying proteins that bind to core origins (reviewed in [20
]). This progress provides grounds for optimism that the trans
-acting proteins that bind to enhancers will also be identified in the relatively near future.