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DNA replication in eukaryotes initiates at multiple origins. The activation of these origins is a critically important event in the life of each cell and is tightly regulated by numerous highly conserved trans-factors.
Saccharomyces cerevisiaeorigins (called Autonomously Replicating Sequences, ARSs) contain a core Aelement called ACS(ARSConsensus Sequence), plus an array of auxiliary Belements. Most ARSsfire at their chromosomal positions, but there are numerous dormant ARSsas well. Instead of being origins, these dormant ARSsserve as silencerelements, which function in the epigenetic repression of nearby genes. Even more, many DNA replication trans-factors have also been reported to affect gene silencing. This puzzling functional duality of ARSand DNA replication factors has attracted significant interest. Evidence from other species has suggested that the overlap between gene silencing and DNA replication operates in other eukaryotes. In this chapter we will review in detail the activity of ARSsas origins of replication and as silencers. We will focus on sequence dissimilarities between silencer and origin ARSsand will propose a model for the functional duality of DNA replication factors.
1.1. Origins of DNA replication in S.cerevisiae
Eukaryotic origins of DNA replication display a significant inter-species diversity. In higher eukaryotes this diversity reaches a point where origin locations are difficult to identify by homology search (Mechali, 2010). A remarkable exception of this diversity occurs in the yeast S.cerevisiae.In this organism the first functional origins have been identified by screens for DNA elements which confer DNA replication on plasmids (Stinchcomb et al., 1979; Chan & Tye, 1980; Kearsey, 1983). Comparison between these autonomously replicating sequences (ARSs) have shown that they encompass approximately 200bp of DNA and contain perfect or one-base mismatches to the 11 bp ARSconsensus sequence (ACS) 5’-WTTAYRTTTW-3’ (where W=A/T; Y=C/T; R=A/G). Linker scanning substitutions in several ARSshave determined that the ACSis the sole essential element for DNA replication (Marahrens & Stillman, 1992; Rao et al., 1994; Lin & Kowalski, 1997; Chang et al., 2008). However, auxiliary Belements (B1-B4) within ARSsare also necessary for full origin activity (Marahrens & Stillman, 1992; Lin & Kowalski, 1997). The B2, B3and B4elements are not present in all ARSs. B2is a site for the unwinding of DNA while B3is a binding site for Abf1p (ARS-binding factor 1), which is a protein involved in numerous chromatin-associated functions including DNA replication, gene silencing, transcriptional activation and DNA repair (Rehman & Yankulov, 2009). The function of B4is unknown, but its mutation reduces replicator activity (Lin & Kowalski, 1997). Interestingly, the destruction of more than one of the Belements substantially reduces origin firing activity (Marahrens & Stillman, 1992) and ACSalone is not sufficient to confer replicator activity at natural yeast chromosomes (Raghuraman et al., 2001).
The B1element, along with the ACS, is found in all known ARSsand forms a bipartite binding site for the Origin Recognition Complex (ORC) (Rao & Stillman, 1995). However, the B1sequence is not nearly as conserved as ACS. The cross-ARShomology at the putative position of B1has been identified as a WTW motif found 17-19 bp upstream of the ACS(Chang et al., 2008) or an AWnY (W=A/T; Y=C/T; n=any nucleotide) motif 16 bases upstream of ACS(Palacios DeBeer et al., 2003). Even more, the whole region upstream of ACSis A/T rich thus providing multiple nearby WTW/AWnY motifs. Ultimately, the precise position of B1and its significance remains somewhat elusive.
1.2. ARSsinitiate replication
A wealth of information has been accumulated on the mechanisms by which ARSsinitiate DNA replication (Fig. 1). ORC, which is built of six different Orc proteins, binds the ACS-B1elements to nucleate the formation of the pre-replicative complexes (Blow & Dutta, 2005; Labib, 2010). Shortly after mitosis, Cdt1p and Cdc6p recruit the heterohexameric MCM complex to the ARS-bound ORC. Thus, ARSsare poised to initiate DNA replication upon receiving a regulatory stimulus. This stimulus is provided in S-phase by two protein kinases, DDK and CDK2 (Labib, 2010). It seems that the critical event in the stimulation of origins is the phosphorylation of Mcm4p (Sheu & Stillman, 2010) by DDK. However, other components of the pre-initiation complex are also phosphorylated with similar timing (Labib, 2010). These events culminate in the activation of the MCM helicase, in the unwinding of origin DNA and in the assembly of the DNA replication machinery.
There are about 12 000 matches or near-matches to ACSin the genome of S. cerevisiae (Nieduszynski et al., 2006). Of these, only 500-700 are loaded with ORC and MCM proteins (Wyrick et al., 2001) and only about 400 initiate DNA replication (Raghuraman et al., 2001). In general, the early firing origins are located in the central portion of the chromosomes, while the later firing origins are found at the periphery (Raghuraman et al., 2001). It is believed that the same initiation events take place at all origins of DNA replication, but at different times throughout S-phase. Interestingly, the dormant origins positioned in the immediate subtelomeric regions or at the mating type loci (see below) also recruit the ORC and the MCM complexes (Wyrick et al., 2001; Rehman et al., 2006), but seldom if at all fire. It is not known how DDK, CDK2 and other initiation factors are regulated to confer the temporal pattern of origin firing, how they discriminate dormant origins or how all these events are coordinated.
The disparity between loading and firing of ARSsin S.cerevisiaeis reminiscent to the situation in metazoans, where tens of thousands of genomic positions are primed as origins, but only a small subset actually fire. It is believed that this excess of available origins can accommodate the significant differences in growth conditions during metazoan development as well as the substantial variation in chromatin structure in different cell types (Mechali, 2010). For example, local chromatin structure, transcription and/or different environmental and physiological conditions will contribute to the selection of the most suitable origins. In this way, unnecessary interference with gene expression or the disturbance of established heterochromatin domains will be avoided.
The so-called Jesuit model (“For many are called, but few are chosen” (Matthew 22:14, the Bible)) has been proposed to explain the limited firing of origins. This model implicates that the considerable flexibility of DNA replication programs is most likely controlled by the abundance of pre-replicative complex factors (such as ORC and MCM2-7) and a corresponding limitation of initiation factors (such as Cdc45, Cdc7p and CDK2) (DePamphilis, 1993). Budding yeast provides an interesting twist to this model. Not only are certain origins chosen to fire while others are not, but some of the non-firing origins aqcuire a completely new role and contribute to the local silencing of genes.
1.3. ARSsact as silencers
Eukaryotic genes are regulated by a variety of mechanisms including complete silencing viacondensed heterochromatin structure. The condensed/relaxed chromatin structures are faithfully transmitted to daughter cells thus ensuring the continuity of gene expression programs. This intriguing epigenetic phenomenon has been extensively studied at the mating type (HMRa and HMLα) (Fig. 2) and at the telomeric loci (Fig. 3) of S.cerevisiae.At all these loci the critical role in gene silencing is played by the SIR (Silent Information Regulator) proteins (Rusche et al., 2003). Through contacts with DNA-binding proteins, Sir1p, Sir3p and Sir4p recruit the Histone-Deacetylase Sir2p. In turn, Sir2p deacetylates the tails of H3/H4 histones on the nearby nucleosome. Additional Sir3p and Sir4p then associate with the deacetylated histone tails to recruit more Sir2p and expand the domain of deacetylated nucleosomes. Ultimately, the deacetylation of histones culminates in the establishment of compacted heterochromatin, which suppresses gene expression. The spreading of SIR proteins is countered by Histone-Acetyl-Transferases and other factors, whose identity and modes of action are not so well understood (Lafon et al., 2007; Ehrentraut et al., 2010).
The silent mating type loci, HMRa and HML, are constitutively and completely repressed by robust heterochromatin structure. The genes encoded by these loci are expressed only when translocated to the MATlocus (Rusche et al., 2003). In turn, the MATlocus can accomodate and express only the a or the genes. In this way, it is gauranteed that no erraneous expression of the two opposing mating types occurs.
HMRa and HMLare each flanked by Eand Isilencers (Fig. 2). Remarkably, ARSshave been identified as essential elements in all four silencers of the these loci (Abraham et al., 1983; Broach et al., 1983; Rusche et al., 2003). For example, the HMRa-Esilencer contains ARS317as well as binding sites for Rap1p and Abf1p, whereas the HMRa-Isilencer contains ARS318and an Abf1p binding site (Fig. 2). Depending on the genomic context, both Abf1p and Rap1p bind to gene silencers or activator elements (Shore & Nasmyth, 1987; Shore et al., 1987). Just as in replication origins, ORC binds to the bipartite ACS-B1of the ARSsin the mating type loci silencers. However,instead of recruiting replication machinery, the Orc1p subunit of ORC recruits Sir1p, while Rap1p and Abf1p bind and recruit Sir3p and Sir4p. As shown in Fig. 2, Sir1p, Sir3p and Sir4p recruit Sir2p to establish a focal point of silencing and initiate the spreading of the SIR proteins. Similar events take place at ARS318in the HMRa-Isilencer.
ARSsalso play a somewhat similar silencing role at the telomeres (Fig. 3). At these loci, the telomeric repeats act as the principal silencers while ARSshave a silencer-enhancing role (Fourel et al., 2002). The telomeric TG1-3 repeats provide multiple binding sites for Rap1p. Similar to the mating type loci, Rap1p recruits Sir3p and Sir4p to establish the initiation point for the SIR protein spreading (Fourel et al., 2002; Rusche et al., 2003). ARSsand Sir1p are not required for this step. However, the absence of subtelomeric ARSsor Sir1p significantly reduces the span of the silenced domain and its stability while the artificial tethering of Sir1p to the telomere boosts the silencing of nearby genes (Chien et al., 1993). Thus, subtelomeric ARSsand their ability to recruit Sir1p through Orc1p play an important, yet secondary role in gene silencing at the telomeres. At other locations, isolated ARSsdo not induce gene repression, but can boost the activity of an existing silencer. For this reason they were classified as proto-silencers (Fourel et al., 2002).
The complexity of telomeric silencing does not end there. Besides ARSs, the repetitive Core Xand Y'elements in the sub-telomere also contain isolated Rap1p and Abf1p binding sites. All these act as weak multiple proto-silencers. In addition, the Core Xand Y'elements harbour anti-silencer modules called sub-telomeric anti-silencing regions (STARs) (Fourel et al., 1999; Fourel et al., 2004; Power, 2011). The combined assembly of proto-silencers and weak anti-silencers produces a multitude of variations in the strength, stability and spreading of telomeric silencing (Fourel et al., 2004). Even more, Core Xand Y’elements contain isolated clusters of telomeric TG1-3 repeats and are able to interact with the telomeres forming t-loop and D-loop structures. The folding back of telomeric DNA brings the SIR
proteins bound to the telomeric repeats into close proximity with those bound to the subtelomeric Core Xelement. This interaction creates a highly condensed heterochromatic structure in a specific region of the sub-telomere while the stretch of DNA between the Core Xand the telomere may actually be euchromatic. The formation of these fold-back structures generates discontinuous telomeric silencing and strong silencing domains can be formed many kilobases away from the telomere (Pryde & Louis, 1999; Fourel et al., 2004). Importantly, as any other ARSs, telomeric proto-silencer ARSsalso contain a normal ACS-B1module, bind ORC and act as origins when placed on mini-chromosomes (Wyrick et al., 2001; Rusche et al., 2003; Chan & Tye, 1980).
So, what confers the functional plasticity of ARSs? The fair answer is that we do not really know. Many studies have correlated the efficiency of origins to their proximity to heterochromatin (Weinreich et al., 2004; Field et al., 2008; Mechali, 2010). Indeed, origins in compact chromatin tend to fire less frequently than origins in open chromatin. It is conceivable that open chromatin is necessary for the assembly of the pre-replicative complexes (Doyon et al., 2006; Espinosa et al., 2010). However, chromatin structure is not the only regulator of origin activity. For example, in the mini-chromosome maintenance assay, (which involves the transfer of different origins to a plasmid and examining their efficiency under the same genetic context), it was found that different ARSsfire at different rates and support different levels of DNA replication (Chan & Tye, 1980; Chang et al., 2008). These observations immediately suggest that variation in the sequence of ARSsmust also play a role in the fine tuning of ARSfunction.
The next step was to take highly efficient ARSsfrom euchromatic regions and insert them into heterochromatic regions known to have poor replication initiation efficiency. Consequently, the high efficiency of the ARSswas lost, showing that the genomic context was powerful enough to overcome the effects of the sequence variation (Weinreich et al., 2004). However, a parallel change in the activity of ARSshas been discovered. Not only were the relocated ARSsshowing reduced replication and late firing, they were now boosting the epigenetic silencing of the nearby genes, contrary to their activities in their native chromosomal locations. A fine twist to these phenomena is that the replicator ARSsdid not make perfect silencers (Casey et al., 2008) or proto-silencers (Rehman et al., 2009), reflecting the fact that silencer ARSssometimes do not make perfect replicators (Chang et al., 2008; Palacios DeBeer et al., 2003). It is not clear how the new chromatin environment of the relocated ARSshas contributed to their functional conversions.
This leads us to the question: is there any feature of an ARSthat determines its predisposition to act as a silencer or a replicator? We and others have recently aligned a number of ARSsin search for some correlation between sequence and function. It was possible to delineate a B1element consensus (WTW) from the origins on chromosome III (Chang et al., 2008). However, silencer and telomeric ARSsshowed even lower conservation of this B1element compared to other ARSs(Rehman & Yankulov, 2009). Another line of evidence has previously shown that B1can modulate the affinity of ACS-B1to ORC in vitroand that silencer ACS-B1have higher affinity to ORC (Palacios DeBeer et al., 2003).
We have hypothesised that the mode of ORC association to ACS-B1can ultimately influence how well an ARSwill act as an origin or a silencer. If this is the case, variations in the B1element and its flanking sequences can potentially contribute to the functional conversions of ARSs. For example, if B1causes ORC to acquire a specific conformation and higher affinity, ORC could end up recruiting the silencing machinery through the Orc1p-Sir1p interaction. In addition, it is also possible that the orientation of ACS-B1towards another silencer could have an impact on the conformation of ORC, again promoting epigenetic silencing rather than replicator function. In both situations, ACS-B1should provide for a significant level of flexibility of ORC, which in turn should allow the acquisition of silencer/replicator function depending on the chromatin context. In the following sections we will present our on-going studies that are testing these models.
2.2. Destruction of B1has different effects in silencer and replicator ARSs
Initial assessment of the role of the B1element has been performed on two well characterised ARSs, the replicator ARS1and the telomeric proto-silencer ARS319(Fig. 4). We inserted these origins with an adjacent URA3reporter in the left telomere of chromosome VIIand assessed the level of repression by a routine assay for the sensitivity of cells to FOA (5-Fluoro-Orotic Acid). FOA is a neutral substance, which is turned into a toxin by the URA3-encoded Orotidine-5'-phosphate-decarboxylase. Hence, cells with repressed URA3will grow in the presence of FOA, while cells expressing URA3will be sensitive to FOA. After transforming with the integrating constructs, cells were selected on media without uracil (SC-ura) and telomeric integration was confirmed by PCR. The transformed cells were then grown in non-selective media for 15-20 generations to reach equilibrium of epigenetically repressed and transcribed URA3and then plated on non-selective plates and plates containing FOA. The proportion of cells with repressed URA3(%FOAR) was assessed as the number of colonies on plates containing FOA (SC+FOA) divided by the number of colonies on non-selective plates. The difference in %FOAR values is indicative of the difference in the levels of silencing at the analysed locus.
These analyses revealed that the destruction of the B1element (TTTccT) in ARS1moderately reduced the %FOAR values suggesting that this B1element contributes to the overall gene silencing at telomeres (Fig. 4). Exactly the same mutation has also significantly reduced the replicator activity of ARS1(Marahrens & Stillman, 1992). Surprisingly, the destruction of the putative B1element (ATTccT) of ARS319had very little effect on telomeric silencing (Fig. 4) and only moderately reduced the replicator activity of ARS319(Chang et al., 2008). These puzzling results suggest that ARS319does not possess an ordinary B1element. It is possible that B1in ARS319is offset from the customary position found in other ARSs.Alternatively, ARS319has a broader B1element that is not affected by the replacement of only two nucleotides.
2.3. Scanning mutations of the B1element in a silencer ARSshow little effect on its silencing and replicator activity
ARS317is a well-characterised core component of the HMRa-Esilencer. Similar to the proto-silencer ARS319, mutations in the putative B1element (TTATcc) of ARS317have little effect on its replicator activity (Chang et al., 2008). In order to assess the role of the ARS317-B1element in gene silencing, we performed a two-nucleotide substitution scanning mutagenesis of the region encompassing its putative B1(Fig. 5). All ACS-b1mutants were cloned next to URA3and inserted in the left telomere of chromosome VIIas before. The level of repression of URA3was assessed by the FOA sensitivity assay as described in the previous section. The results indicated that the destruction of the WTW motif (TTAccA or TTATTc) did not reduce, but actually slightly increased the silencing of URA3(Fig. 5). The only moderate decrease in silencing was observed in the construct 1.5, where a GC pair proximal to ACSwas replaced with an AA (GCaa). This result is somewhat surprising as G/C bases do not conform to the general A/T rich nature of this region. In conclusion, the canonical B1elements of both ARS317and ARS319seemed dispensable for silencer function (Fig. 4 and Fig. 5) and had little or no effect on the replicator activity of these ARSs(Chang et al., 2008).
2.4. Scanning mutations of the B1element of a replicator ARSaffect both silencer and replicator function
We conducted a similar scanning mutagenesis analysis of the B1element in one of the most active origins of DNA replication in the genome of S.cerevisiae, ARS305(Huang & Kowalski, 1996). Two-nucleotide substitutions were introduced at the positions shown in Fig. 6 and the mutant ACS-b1constructs were attached to URA3.These reporter cassettes were inserted in the left telomere of chromosome VIIand analysed for the levels of URA3repression. The analysis showed that, similar to ARS1, the destruction of B1in ARS305reduced the levels of gene silencing at the VII-Ltelomere (Fig. 6). In Fig. 6, it is interesting to note that the 1.1 construct (substitution of one base from the WTW motif and one adjacent base) did not reduce the silencing ability while the 1.2 construct (substitution of two bases in the WTW motif) caused silencing ability to decrease by approximately 15%. Also, it is curious that mutations in the WTW flanking sequences (constructs 1.3 and 1.5) showed the greatest
reductions in silencing (30% and 20% reductions of %FOAR respectively). Very similar mutations have been shown to affect the activity of ARS305as a replicator (Huang & Kowalski, 1996). So, the two replicator ARSswe have analysed possess a well preserved B1element, which functions in both origin firing and in epigenetic silencing. Also, scanning substitutions of the ARS305B1element revealed that mutations in the B1flanking sequences have significant effects on gene silencing as well. In contrast, the two silencer ARSswere unaffected by any of the two-nucleotide substitutions in the B1region.
2.5. Is there any substantial difference to B1in replicators and silencers?
The subtle differences in the activities of B1elements in select replicator and silencer ARSsprompted us to perform extensive sequence alignments of multiple ARSsaccording to their function and/or location in the genome. ARSswere grouped as replicators (ARSsthat are located away from the telomeres and the silencer loci, which confer autonomous replication when moved to a plasmid), silencers (ARSsfrom the HMloci and the rDNAlocus) and ARSswithin 5 kb of the telomeres. We note that the latter category contains ARSsthat are imbedded in the repetitive Core Xand Y’subtelomeric elements (Chan & Tye, 1983; Walmsley et al., 1984) and that they share higher homology in the sequences outside the ACS. All sequences were imported in WebLogo (www.weblogo.berkeley.edu) and analysed for similarities (Fig. 7).
These analyses confirmed the higher sequence conservation in the vicinity of replicator B1elements that was reported earlier (Chang et al., 2008). Nevertheless, telomeric proto-silencer and silencer ARSsseem to contain broader WTTTTT and WTTT consensus sequences, respectively, as compared to the WTW consensus of the replicators. These slight variations corroborate the differences observed in the scanning mutation analyses of ARS305(replicator) and ARS317(silencer). It is quite possible that the broader A/T rich stretch in B1of the silencer ARSscontribute to the lower effect of the two-nucleotide substitutions in ARS317as compared to ARS305(Figs. 5 and 6). However, we need to stress that both the effects in the silencing assays and the difference in the B1sequence are subtle and do not really reveal a major feature that can distinguish between the two types of ARS.
2.6. The orientation of ACS-B1determines the levels of telomeric gene silencing
Previous studies have indicated that the orientation of the HMRa and HMLsilencers impose directional repression of genes (Zou et al., 2006a; Zou et al., 2006b). In particular, the HML-Iand the HMR-Esilencers were found to more efficiently repress URA3reporters if oriented B1-ACS-Rap1-Abf1-URA3(Fig. 2) (Zou et al., 2006b). These effects were linked to the ability of ACS(and supposedly ORC) to robustly position a nucleosome towards the Abf1side of the silencer (Zou et al., 2006a). Towards the B1side of ACSthere is no stably positioned nucleosome and the silencing of URA3is significantly weaker. Interestingly, the replicator ARS1has a stably positioned nucleosome at both the B1and the Abf1sides of ACS(Lipford & Bell, 2001; Zou et al., 2006a).
It is not entirely clear what determines the directional effects of the HML-Iand HMR-Esilencers. For example, do Abf1p and Rap1p (and presumably other proto-silencers) facilitate the rigorous control of the nucleosome positions or does ACS-B1/ORC work independently? In this line of thought, many origins (such as ARS305, ARS307, ARS605) do not contain Abf1p or Rap1p binding sites, while in others (ARS1, ARS319) B1and the Abf1p binding site reside on the same side of ACS(Marahrens & Stillman, 1992; Rao et al., 1994; Huang & Kowalski, 1996; Rehman et al., 2009). Invariably, all these ARSsimprove gene silencing when inserted at the telomere ((Rehman et al., 2009) this article). We decided to test if these ARSsalso display directional silencing.
Initial experiments were conducted using ARS605and ARS319. As mentioned earlier, ARS605has no apparent Abf1p binding site, while in ARS319both B1and the Abf1p binding sites are at the same side of ACS. We have cloned these origins in both orientations relative to URA3and the telomere to produce the URA3-ACS605-B1-tel, URA3-B1-ACS605-tel, URA3-ACS319-B1-teland URA3-B1-ACS319-telconstructs. These constructs were inserted in the left telomere of chromosome VIIand the levels of URA3repression were assessed as before (Fig. 8).
Our results indicate that both ARS319and ARS605boost the repression of URA3in the URA3-ACS-B1-telorientation. These results are in tune with earlier observations (Zou et al., 2006a; Zou et al., 2006b). Very interestingly, in the opposite direction these ARSsmarkedly reduced the repression of URA3(Fig. 8). So, similar to HML-I (ARS302)and HMR-E (ARS317), ARS605and ARS319display directional silencing, but also act as anti-silencers in the opposite direction. Assuming that a similar robustly positioned nucleosome next to ACSdetermines the direction of silencing of ARS605and ARS319, we argue that the lack of a stable nucleosome at the B1side of these ACSscan serve as an insulator against the spreading of SIR proteins from the telomere and dampen silencing. We also suggest that other ARSsincluding ARS302and ARS317will have a similar insulating activity. Together, our results indicate that the orientation of ORC towards a nearby silencing domain (such as the telomere or the HMloci) has a significant impact on the strength of silencing.
3.1. Role of the B1element in ARSduality
The central topic of the presented studies is the enigmatic dual function of ARSsas replicators and as silencers. Because earlier studies have shown distinct affinity of ORC to ARSsin silencers and replicators and because B1has been proposed to affect ORC affinity (Palacios DeBeer et al., 2003), we have focused on the role of this element on gene silencing at the telomeres. We have compared our results to similar analyses on the role of B1in origin activity.
We have found that mutations in the B1elements of replicator ARSsreduce their activity in telomeric silencing (Figs. 4 and 6). Similar mutations have also reduced the replicator activity of these ARSs(Chang et al., 2008; Marahrens & Stillman, 1992). So, replicator ARSsseem to have a well defined B1that is important, but not required, for both replication and silencing. However, mutations in the B1elements of silencer ARSsseem not to affect silencing (Figs. 4-5). Similarly, mutations in the B1of these ARSshave a lesser effect on replication activity than the effects seen in replicator ARSs(Chang et al., 2008; Marahrens & Stillman, 1992). This leads us to the hypothesis that silencer ARSshave a special type of B1element. In this line of thought, we have also noticed that mutations in the sequences flanking the WTW motif in replicator ARSsaffect silencing to a greater extent than silencer ARSs(Fig. 6). This observation suggests that the sequences flanking WTW are more important for silencing than for replication and argue in favour of a broader B1element in silencer ARSs.
We propose that the subtle functional differences between silencer and replicator ARSsis due to the broader B1consensus sequence in silencers. We suggest that silencer ARSscontain more A/T base pairs around the WTW motif. Support for this hypothesis was provided by the alignment of different types of ARSs. In Fig. 7, we introduced the notion of wider B1elements, where a consensus of WTTTTT was found for proto-silencer ARSsand WTTT was found for silencer ARSs. Replicator ARSsshowed only the previously described WTW motif (Chang et al., 2008). It is possible that ARSswith broad B1elements would be more accommodating to mutations within the consensus because the adjacent bases would still resemble a WTW motif. The adjacent WTW sites may be able to act as alternative sites for the attachment of ORC (Fig. 9). It is also possible that these ARSspossess additional B1elements that render the mutations in WTW insignificant.
Earlier structure-function analyses of the association of ORC to ARS1(Rao & Stillman, 1995) have indicated that ORC binds to both ACSand B1(Fig. 9). Through cross-linking studies, a third minor position of ORC contact with DNA (depicted by “nnn” in Fig. 9) has also been revealed in-between ACSand B1(Rao & Stillman, 1995). This third site has never been shown to influence the activity of ARS1, but could be important for fine conformational variations in ORC. On the other hand, the small effects of B1in replicator and silencer assays stress its auxiliary nature. Whereas ACSis required for interaction with ORC, the precise roles of B1and the “nnn” sequences remain elusive. It is possible that these auxiliary sequences are adaptor elements, which modulate alternative structures of ORC (Fig. 9).
We can imagine that some of these alternative structures would not expose Orc1p, reducing its interaction with Sir1p, and thus promoting replicator activity instead of silencing. Other conformations of ORC, which expose Orc1p, would stimulate its interaction with Sir1p and increase the ARS’ssilencer activity. The broader B1element present in proto-silencer and silencer ARSsmay allow ORC more binding flexibility than the narrower WTW in replicator ARSs. This increased flexibility may result in greater variations in ORC conformation, thus providing more opportunities for Orc1p to be exposed. A broader B1can also provide additional sites for the attachment of ORC and in turn increase the affinity of ORC to ACS-B1. Such a scenario can explain the previous observations on the link between ORC affinity and stronger silencing. In particular, it has been discovered that strong ORC-DNA interaction at HMRa increased heterochromatin formation and decreased and delayed the initiation of DNA replication. Conversely, weak ORC-DNA interaction caused earlier and increased replication initiation and decreased the formation of heterochromatin (Palacios DeBeer et al., 2003).
Very importantly, alterations in B1by no means eliminate the dual nature of ARS. Whereas broad B1elements seem to prevail in silencer and proto-silencer ARSs(Fig. 7), many replicator B1elements reside in an A/T rich environment as well. This environment can also supply alternative sites for ORC binding. In summary, the B1elements seem to unveil a minor difference between replicators and silencers, but this difference is not strong enough on its own to determine the function of an ARS.
3.2. Flexibility of ORC and the role of chromatin in ARSduality
The orientation of ACS-B1towards a potent silencer seems to be more important than the nature of B1. In support, we (Fig. 8) and others (Zou et al., 2006a; Zou et al., 2006b) have found that the orientation of ARSstowards a powerful silencer such as the telomere or the HMloci can significantly contribute to gene silencing. These effects suggest that ORC is highly flexible since a simple switch of direction contributes so significantly to silencing and anti-silencing. An interesting experiment would be to test how the replicator activity of ARSsis affected based on its orientation towards a silencer.
It is well known that the origin activity of an ARSis governed by chromatin structure (Weinreich et al., 2004). As mentioned previously, both replicator and silencer ARSsact as functional origins of replication when placed on plasmids (Chan & Tye, 1980). Similarly, both replicator and silencer ARSsact as functional silencers and proto-silencers in the HMloci and at the subtelomeres, respectively (Casey et al., 2008; McNally & Rine, 1991; Weinreich et al., 2004). Since an ARS’slocation within the genome determines its function regardless of its original silencer/replicator classification, it appears that the sequence of the ARSsis of lesser importance than the genomic context. Hence, it seems that the communication of ORC with heterochromatin is more important than the way ORC interacts with ACS-B1.Again, ORC is posing as a highly flexible complex, this time in regards to its interaction with chromatin.
Our ideas that the flexibility of ORC can be influenced by chromatin feed some thought on how origins are chosen as per the Jesuit model. In metazoans, the positions of potential origins and origins that actually fire will vary depending on the epigenetic state of the genome. It makes sense not to fire origins that will disturb heterochromatin. One of the ways to ensure that this does not happen is to force the key regulatory factor for origin activation (this being ORC) to function in a different mode. This way, though ORC interacts with all of the potential origins, it will recruit replication machinery only if it is in a euchromatic region. If ORC is bound to DNA that lies within a heterochromatic region, the heterochromatin dictates that ORC will only recruit silencing machinery and maintain the heterochromatic state instead of stimulating replication.
Despite our extensive efforts, we have not identified a clear and strong distinctive feature for the replicator or the silencer ARSs. This brings us back to the idea that ARS,together with the associated ORC and other DNA replication factors, is a flexible bi-functional module that can be remoulded depending on the chromosomal context and perhaps by additional factors such as the need of the cell to divide or to modulate its gene expression. In other eukaryotes we see no readily identifiable consensus sequence in origins of DNA replication (Mechali, 2010). Even so, strong links of ORC to gene silencing and heterochromatin have been identified by many studies in Schizosaccharomyces pombe and in higher eukaryotes (Pak et al., 1997; Auth et al., 2006; Deng et al., 2007; Stuermer et al., 2007; Kato et al., 2008; Deng et al., 2009; Prasanth et al., 2010). Perhaps ORC has evolved to lose its stringent sequence requirements for binding to DNA, but the origin-ORC module has maintained its flexibility and the ability to accommodate varying conformations.
It is somewhat anecdotal that while looking for updates on the Jesuit model for the firing of metazoan origins (DePamphilis, 1993) we came across this citation dealing with the duality of the universe: "...Light and Darkness, Life and Death, Right and Left.... are inseparable.... For this reason each one will dissolve into its earliest Origin..." (The Gospel of Philip, New Testament Apocrypha). It seems that duality has been encripted in the earliest origin and then preserved through evolution.
The studies in this article have been supported by grants to K.Yankulov from NSERC. D. Jeffery has been supported by studentships from OGSST and from NSERC. P. Chisamore-Robert is supported through stipend from the University of Guelph and NSERC.
Patricia Chisamore-Robert, Daniel Jeffery and Krassimir Yankulov (August 1st 2011). The Silencing Face of DNA Replication: Gene Repression Mediated by DNA Replication Factors, DNA Replication - Current Advances, Herve Seligmann, IntechOpen, DOI: 10.5772/19551. Available from:
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