Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

Authors


E-mail lucia.fabiani@uniroma1.it; Tel. (+39) 6 4991 2243; Fax (+39) 6 4991 2351.

Summary

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.

Introduction

The replicator sequences required for eukaryotic origins were first identified in Saccharomyces cerevisiae as chromosomal DNA fragments capable of promoting high-frequency transformation and the autonomous replication of plasmids. They are called autonomously replicating sequence (ARS) elements. They are modular in structure, consisting of an essential domain A and elements in the B and C domains that stimulate ARS activity (reviewed by Newlon and Theis, 1993; Newlon, 1996). Domain A contains an 11 bp consensus sequence, the ARS consensus sequence (ACS), that acts as the core of the recognition sequence for the replication initiator protein, the six-subunit origin recognition complex (ORC). The ACS and element B1, localized 3′ to the T-rich strand of the ACS, contribute to ORC binding. ORC serves as the ‘landing pad’ for the recruitment of proteins involved in the regulation of the initiation of DNA replication (reviewed by Bell and Dutta, 2002).

Saccharomyces cerevisiae is an excellent model for eukaryotic DNA replication, but its replicators appear to be different from replicators in other eukaryotes. These replicators are larger, varying from a few hundred basepairs in some unicellular organisms, e.g. Schizosaccharomyces pombe, Physarum polycephalum and Yarrowia lipolytica (Bénard et al., 1996; Dubey et al., 1996; Vernis et al., 1999), up to several kilobasepairs in mammals (Aladjem et al., 1998). The analysis of S. pombe replicators revealed that they are 0.5–1.5 kb in length and are composed of multiple redundant elements important for ARS function (Kim and Huberman, 1998; 1999; Okuno et al., 1999). Recent results indicate that at least part of the redundancy results from the presence of multiple binding sites for ORC (Kong and DePamphilis, 2002; Takahashi et al., 2003), making S. pombe replicators analogous to S. cerevisiae compound replicators, which contain multiple matches to the ACS, each of which must be altered to abolish replicator activity (Theis and Newlon, 2001). Replication initiation events in some mammalian replication origins appear to be distributed through large ‘initiation zones’ (reviewed by DePamphilis, 1999). Despite the differences in origin structure among different organisms, the conservation of replication initiation proteins, including ORC, from yeast to mammals suggests a common feature in origin structure (reviewed by Bogan et al., 2000).

The yeast Kluyveromyces lactis provides a good system for the study of chromosomal replicators (Fabiani et al., 1990). We showed previously that a 103 bp fragment containing KARS101 was a replicator in both K. lactis and S. cerevisiae. This fragment contains a 12 bp sequence that appears to function as the essential ACS in S. cerevisiae, but it only contributes to ARS activity in K. lactis. Further analysis of KARS101 revealed that the essential core of KARS101 is 40 bp in length, at least twice as large as the essential core of S. cerevisiae ARS elements, and that different sequences are required for ARS function in the two organisms (Fabiani et al., 1996). We recently identified in the circular plasmid pKD1, which is able to transform K. lactis and S. cerevisiae, a 159 bp sequence that promotes autonomous replication in both yeasts. This fragment contains a sequence related to the S. cerevisiae ACS and a region of 53% identity to KARS101 essential core. Examination of pKD1 replication intermediates by two-dimensional electrophoresis revealed a correlation between the ARS element and the origin of DNA replication (Fabiani et al., 2001).

In this paper, we analyse KARS101 replication intermediates to examine its role as an origin of DNA replication in its chromosomal context. In addition, we define the cis-acting sequences required for K. lactis origin function by substitution at the chromosomal level of the KARS101 wild-type sequence with a mutated copy having a complete deletion of the core previously shown to be essential for K. lactis ARS function (Fabiani et al., 1996). We also demonstrate, by in vitro analysis, that the sequence essential for origin function is the binding site for protein(s) that are likely to be involved in the initiation of DNA replication.

Results

KARS101 is an efficient chromosomal origin of DNA replication

Kluyveromyces lactis ARS sequence, KARS101, is an efficient replicator in plasmids (Fabiani et al., 1990; 1996). Figure 1 shows the sequence of the 103 bp KARS101 ClaI–SspI fragment that has ARS activity in S. cerevisiae and K. lactis, as well as the elements previously shown by deletion and linker scanning mutagenesis to contribute to ARS function. The dodecanucleotide is essential for ARS activity in S. cerevisiae. The 40 bp core sequence essential for K. lactis ARS function (Fabiani et al., 1996) is within a 50 bp region conserved in three K. lactis ARS elements, including a chromosomal ARS element (KARS12) and the replication origin of the plasmid pKD1 (Fabiani et al., 2001). To define the function of KARS101 as a chromosomal origin of DNA replication, we analysed the replication intermediates of KARS101 by two-dimensional electrophoresis techniques (Brewer and Fangman, 1987; Huberman et al., 1987). The first step was to define the chromosomal restriction map of the region containing KARS101. K. lactis chromosomal DNA was digested with various restriction enzymes, used separately or in combination. The physical map deduced from this analysis is shown in Fig. 2.

Figure 1.

Schematic representation of the 913 bp KARS101 HindIII–PstI fragment that replicates efficiently in K. lactis and S. cerevisiae. KARS101 was initially identified as a 913 bp HindIII–PstI fragment. The 103 bp ClaI–SspI subclone replicates in K. lactis and S. cerevisiae. The boxes on the sequence indicate the dodecanucleotide required for ARS activity in S. cerevisiae and the 40 bp core essential for ARS activity in K. lactis.

Figure 2.

Chromosomal map and two-dimensional gel analysis of replication intermediates of KARS101. The restriction map of the chromosomal region containing KARS101 is shown at the top. The map was defined by Southern analysis of genomic DNA, using the 913 bp KARS101 HindIII–PstI fragment as probe. The box represents the 103 bp sequence, and the black box inside represents the essential core. DNA for two-dimensional analysis was isolated as described in Experimental procedures. The first dimension was run in 0.4% agarose gel at 1.2 V cm−1 for 28 h, and the second dimension was in a 1% agarose gel at 5 V cm−1 for 10 h.
A. Schematic diagram showing the replication intermediates detected after hybridization.
B, C and D. The two-dimensional patterns obtained after digestion with EcoRI–XbaI (B), PvuII (C) and EcoRI–XhoI (D). The blots were probed with the 913 bp HindIII–PstI fragment described above.

DNA was prepared from exponentially growing cells, and the replication intermediates (RIs) were analysed by two-dimensional gel electrophoresis. The first dimension separates the fragments according to their size, and the second according to their size and structure. We digested K. lactis chromosomal DNA with appropriate restriction enzymes to obtain 3–5 kb fragments containing KARS101 in different relative positions. If replication initiates at or near KARS101, we expected to see bubble-shaped RIs of restriction fragments in which the ARS element is near the centre of the fragment. Consistent with this expectation, the RIs of the 5.7 kb EcoRI–XbaI fragment and the 4.3 kb PvuII fragment formed clear arcs of bubble-shaped RIs. In both cases, the patterns were discontinuous, with a strong signal produced by large Y-shaped RIs (Fig. 2B and C). This discontinuous pattern can be explained by forks moving bidirectionally from the ARS element, located ≈ 35% of the distance from one end of the fragment to the other. If the two forks move at equal rates, then RIs should be bubble-shaped for the first 70% of replication and Y-shaped for the remainder. These two patterns and others not shown suggest that KARS101 is an efficient origin of DNA replication. The pattern of Y-shaped RIs seen in the analysis of the 4.8 kb EcoRI–XhoI fragment, which has KARS101 close to one end, provided further support for the idea that replication initiates at or near KARS101 (Fig. 2D). In this case, the RIs are expected to be predominantly Y-shaped because replication initiates so close to the end of the fragment.

Essential role of the core sequence in KARS101 origin function

After the demonstration that KARS101 was an efficient origin of DNA replication in its chromosomal location, we wished to define the sequences that are essential for K. lactis replicator function. We deleted from the chromosome a 50 bp sequence containing the essential core of KARS101 to test its role in replicator activity (see Fig. 1). For this purpose, we isolated from a K. lactis genomic bank a plasmid, pCC10, carrying a KARS101 fragment of about 6 kb (Fig. 3A) that included the EcoRI and XbaI sites shown on the map in Fig. 2. This fragment was large enough to facilitate the homologous recombination necessary for the substitution of the wild-type KARS101 sequence with the deletion mutant.

Figure 3.

Isolation of the KARS101 genomic sequence and effect of the insertion of the URA3 gene on replication activity of KARS101.
A. The restriction map of KARS101 genomic fragment present in the plasmid pCC10 identified by colony hybridization. The S. cerevisiae URA3 gene was inserted in the XhoI site.
B. The ARS phenotypes of the 1.3 and 2.4 kb HindIII–BglII fragments without and with URA3.
C. The two-dimensional pattern obtained after digestion with PvuII of chromosomal DNA isolated from the K. lactis strain carrying the URA3 insertion near KARS101. The gel was run and probed as described in the legend to Fig. 2.

We inserted a selectable marker, the S. cerevisiae URA3 gene, in the XhoI site at a distance of 0.7 kb from KARS101 (Fig. 3A). To test whether the URA3 insertion compromised plasmid ARS activity, we subcloned the KARS101 HindIII–BglII fragments with and without the URA3 insertion in the centromere-containing plasmid, pLF1. Both plasmids were able to transform K. lactis efficiently. Therefore, the insertion of URA3 close to KARS101 does not affect its ARS activity (Fig. 3B).

To test whether the URA3 insertion affected the activity of KARS101 at the chromosomal level, we transformed K. lactis with the 5.0 kb PvuII genomic fragment with the URA3 gene inserted in the XhoI site (Fig. 3C). We used Southern analysis to identify a K. lactis transformant carrying URA3 at the correct position, and analysed KARS101 replication intermediates by two-dimensional gel electrophoresis (Fig. 3C). The two-dimensional pattern obtained shows the typical bubble arc, indicating that the KARS101 PvuII fragment is replicated by an internal active origin. Therefore, the URA3 insertion does not compromise KARS101 origin function at the chromosomal level.

Finally, we constructed a plasmid, pLF101, in which the 50 bp region of homology shown in Fig. 1 was deleted from the URA3-marked KARS101 as described in Experimental procedures(Fig. 4A). This plasmid was unable to transform K. lactis at high frequency, demonstrating the essential role of the 50 bp sequence in plasmid ARS activity. The 5.0 kb PvuII fragment obtained from this plasmid was used to transform K. lactis. We used Southern analysis to identify a transformant strain with the KARS101 deletion. Figure 4B shows the two-dimensional agarose gel pattern obtained by analysis of the mutant strain. Deletion of the 50 bp sequence containing the KARS101 core abolished the activity of the replicator, as shown by the absence, after a long exposure, of bubble-shaped RIs. The RIs detected were Y-shaped, indicating that the mutant fragment is replicated by an external origin (Fig. 4B, left). To show that the failure to detect a replication bubble arc in the KARS101 deletion fragment was not a result of loss of replication bubbles from the DNA preparation, we analysed the replication intermediates of another K. lactis chromosomal origin (KARS12), and a replication bubble was readily detected (Fig. 4B, right).

Figure 4.

Map of the KARS101 core deletion mutant and two-dimensional analysis of the mutant K. lactis strain.
A. Map of the fragment used to replace the chromosomal copy of KARS101.
B. The two-dimensional patterns obtained after PvuII digestion of chromosomal DNA isolated from the mutant K. lactis strain carrying a complete deletion of the 40 bp core sequence and URA3 as selectable marker. The filter was first probed with the 913 bp KARS101 fragment described in the legend to Fig. 2 (pattern on the left) and then with the 594 bp PstI–BglII fragment containing KARS12 (pattern on the right). The white boxes along the fragment represent KARS101 and KARS12; the black box inside KARS12 represents the 50 bp sequence with homology to the KARS101 core sequence.

These results demonstrate that sequences essential for chromosomal replicator activity are contained within the 50 bp region containing the core sequence previously shown to be essential for K. lactis ARS activity. In addition, they show that KARS12 is active as a chromosomal replication origin.

Role of the core in protein binding using K. lactis nuclear extract

We hypothesized that the KARS101 core could represent the binding site of a protein complex essential for the initiation of DNA replication in K. lactis. To test this idea, we prepared a nuclear extract from exponentially growing K. lactis cells and analysed, by gel retardation assays, protein binding to the 103 bp KARS101 ClaI–SspI fragment. Using increasing amounts of nuclear extract, containing up to 20 µg of protein, we found that the addition of 10 µg of protein was sufficient to shift completely the band corresponding to 0.5 ng of the KARS101 fragment. Figure 5A demonstrates that the 50 bp fragment containing the essential core of KARS101 competes with the binding. An excess of 100-fold (Fig. 5A, lane 5) was required to abrogate the binding completely. This result was confirmed using as DNA substrate two reassociated synthetic oligos corresponding to the sequence used as competitor.

Figure 5.

Gel mobility shift assays using K. lactis nuclear extract.
A. The 121 bp SalI–BamHI fragment, amplified by PCR using oligo A and B and containing the 103 bp fragment of KARS101, used in the binding reaction and the 50 bp sequence containing the 40 bp essential core used as specific competitor (black area). The panel on the right shows competition assays. Lane 1, no protein added; lanes 2–5 contained a constant amount of nuclear extract (15 µg); lane 2, no competitor; lanes 3, 4 and 5 contained 10, 50 and 100 molar excess of competitor respectively.
B. Gel mobility shift assays using nuclear extract (NE) and different competitors: lane 1, no protein; lane 2, no competitor; lanes 3–9, 50 molar excess of the competitors shown on the right. Specific competitors (SC) were prepared by PCR using wild-type and mutant KARS101 sequences as templates and oligo A and oligo B, indicated in (A), as specific primers. The black area in the 103 bp fragment represents the 40 bp essential core. The sequence b has the deletion of the region between nucleotides 54 and 68; the substitution mutants c, d, e and f have the sequences represented in grey substituted with the BglII linker (Fabiani et al., 1996).

To define better the sequence important for protein binding, we used as competitors KARS101 substitution mutant sequences previously shown to eliminate KARS101 activity in plasmids (Fabiani et al., 1996) (Fig. 5B). Only the wild-type sequence was able to compete efficiently for binding (Fig. 5B, lane 4); no competition was observed using the sequence between nucleotides 1 and 49 (Fig. 5B, lane 3), and only a faint competition was observed using a fragment with a complete deletion of the dodecanucleotide (Fig. 5B, lane 5) or different linker substitution mutations (Fig. 5B, lanes 6–9). Therefore, the intact core sequence is required for efficient binding. These results were confirmed using the mutated sequences as probes.

In Fig. 5A and B, the complex is larger in the lanes lacking competitor DNA (lanes 2) than in other lanes. This increase in size is probably the result of an excess of nuclear extract in the binding reaction. In fact, we have observed a progressive increase in the size of the band corresponding to the DNA–protein complex as we used increasing amounts of nuclear extract (data not shown).

The binding of ORC to S. cerevisiae ARS elements requires ATP, and the ATP-dependent binding of ORC to DNA has also been observed in Drosophila melanogaster (Austin et al., 1999) and Sciara coprophila (Bielinsky et al., 2001). We observed no difference in the binding of proteins to KARS101 in the absence or presence of ATP (data not shown). One explanation for this result is that the amount of ATP in the nuclear extract is sufficient for binding. The K. lactis nuclear extract was loaded on a G25 column to remove the ATP and, after fractionation, we used a fraction enriched in proteins in gel shift mobility assays. Filtration of the nuclear extracts through the G25 Sephadex column almost completely eliminated the free ATP in the extract (data not shown), but not the bound ATP. Using the G25 protein-containing fraction, we observed an increasing yield of the specific DNA–protein complex (marked with a black arrowhead) with the addition of increasing amounts of ATP (Fig. 6, lanes 3 and 4). The partial recovery of binding is likely to reflect a lower concentration of proteins in the G25 eluate. The higher molecular weight band (marked with an open arrowhead) may be due to non-specific binding, which is stronger in the absence of added ATP (Fig. 6, lane 2) and progressively weaker with the addition of ATP (Fig. 6, lane 3 and 4). These results suggest that binding of proteins in the nuclear extract to KARS101 requires ATP.

Figure 6.

ATP dependence of protein binding. A G25 fraction enriched in proteins was used in the gel mobility shift assay. The amount of protein used is one-fifth the amount used in the bandshift assays shown in Fig. 5. Lane 1, no protein added; lane 2–4, a fixed amount of proteins has been added; in lane 2, no ATP; in lanes 3 and 4, ATP was added at a concentration of 1.5 and 3 mM respectively. The black and open arrowheads indicate specific and-non-specific binding respectively.

Role of the dodecanucleotide in protein binding using S. cerevisiae nuclear extract

KARS101 is an efficient origin of DNA replication in S. cerevisiae, and it was interesting to analyse the in vitro binding using S. cerevisiae nuclear extracts (Fig. 7). Using increasing amounts of nuclear extract, we found that extract containing 20 µg of protein was required to shift the KARS101 fragment completely. This amount of protein is at least twice the amount of K. lactis nuclear extract required for a similar saturation. The binding appears to depend on sequences within the 50 bp region, as shown by competition assays using the 50 bp sequence as competitor and the failure of the sequence containing the upstream 49 bp sequence to compete (Fig. 7, lane 3). The presence of the dodecanucleotide is essential for binding; a competitor with a complete deletion of the dodecanucleotide (indicated by ‘b’ in Fig. 7B) was not able to compete (Fig. 7B, lane 4). Similarly, a competitor containing a substitution that partially removes the dodecanucleotide (construct ‘c’) failed to compete (Fig. 7B, lane 5). Sequences 3′ to the dodecanucleotide missing in substitution mutant 68-77 were also essential (Fig. 7B, lane 6). These results were confirmed using the deletion and substitution mutants as probes. The substitution mutant 79-88 competed as effectively as wild-type competitor (Fig. 7B, lanes 7 and 8).

Figure 7.

Gel mobility shift assay using S. cerevisiae nuclear extract.
A. Gel mobility shift assay using 20 µg of S. cerevisiae nuclear extract. Lane 1, no protein; lanes 2–8 contained a constant amount of nuclear extract (20 µg), lane 2, no competitor, lanes 3–8 contained a 50 molar excess of the competitors shown on the right. The black area in the 103 bp fragment represents the dodecanucleotide essential for ARS activity of KARS101 in S. cerevisiae. The substitution mutants, c, d and e, have the sequences represented in grey substituted with the BglII linker (Fabiani et al., 1996).

In vitro footprinting

DNase I footprinting analysis was used to delineate at a higher resolution the binding domain(s) of factor(s) present in the nuclear extract (Fig. 5). The 137 bp fragment was obtained by polymerase chain reaction (PCR) amplification as shown at the bottom of Fig. 8 and, after SalI digestion, the fragment was filled in with Klenow using a radiolabelled precursor. The labelled fragment was incubated with K. lactis nuclear extract as usual, and then different amounts of DNase I were added to get at least one cut per molecule of DNA (Fig. 8A). The DNA–protein complex (r) was then resolved from free fragment (f) by polyacrylamide electrophoresis under the conditions reported in Fig. 5. The retarded bands (r) indicated by circles (Fig. 8A) were eluted from the gel and examined by denaturing PAGE (Fig. 8B). Lane 1 shows DNA from lane 1 of Fig. 8A treated with DNase I in the absence of nuclear extract. An almost continuous ladder of bands is evident, indicating free access of DNase I. Lanes 4 and 6 show samples treated with two different amounts of DNase I, 0.15 and 0.25 units respectively. These samples, as for lane 1, derive from those reported in Fig. 8A, lanes 4 and 6 respectively. A diffuse protection towards DNase I digestion is evident in both samples. Four areas are particularly protected (brackets in Fig. 8B). In order to reveal enhanced/protected regions better, we performed densitometric scanning (Fig. 8C) of lanes 1 and 4 (Fig. 8B). Bars indicate the most protected regions (ratios < 1), and numbers refer to hypersensitive site map positions (ratios > 1). Both the core element and flanking regions are shown to be bound by proteins contained in the nuclear extract, supporting the binding/mutagenesis data reported in the previous sections.

Figure 8.

In vitro footprinting of KARS101.
A. The DNA-binding reaction performed as described previously using the 137 bp fragment amplified as shown in Fig. 5A. It was digested with SalI and labelled with Klenow. The DNA-binding reactions were incubated with different amounts (triangles) of DNase I: 0.1, 0.15, 0.2 and 0.25 units (lanes 3, 4, 5 and 6 respectively) to get at least one cut per molecule of DNA. Lane 1, DNA treated with DNase I in the absence of nuclear extract, lane 2, DNA–protein complex without digestion. The DNA–protein complex (r) was resolved from free fragment (f) by polyacrylamide electrophoresis. The bands free (lane 1) and retarded (lanes 2, 4 and 6) indicated by circles were excised and eluted from the gel and finally analysed by denaturing gel electrophoresis as shown in (B).
B. Lane 1 shows DNA from lane 1 of (A). Lanes 4 and 6 show the samples treated with two different amounts of DNase I, 0.15 and 0.25 units respectively. U shows the untreated DNA from sample 2, lane 2, in (A). M is the size marker (pBR322 digested with MspI). The different protected regions are indicated by brackets, and the hypersensitive sites are indicated as black arrowheads.
C. Densitometric analysis of lanes 1 and 4. Numbers indicate hypersensitive site map positions (ratio > 1), and bars indicate protected regions (ratio < 1). For details, see Experimental procedures. The positions of protection and hypersensitivity are shown on the diagram at the bottom. The core is indicated by the black box.

Discussion

The analysis of the replication intermediates of the ARS element KARS101 presented in this paper demonstrates that KARS101 is an efficient chromosomal replicator. KARS101 is localized on K. lactis chromosome IV. These data confirm and extend the correlation between ARS sequences and origins of DNA replication in K. lactis observed previously in the circular plasmid pKD1 (Fabiani et al., 2001).

The sequence comparison among three K. lactis replicators, two chromosomal, KARS101 and KARS12, and one from the pKD1 plasmid, has shown the presence of a conserved 50 bp region present in all three K. lactis replicators and localized in the regions that we showed to be important for ARS activity. The pKD1 conserved sequence is present in the 159 bp sequence essential for efficient plasmid replication in K. lactis (Fabiani et al., 2001). The 40 bp core sequence of KARS101 (Fabiani et al., 1996) is within the 50 bp conserved region. We show in this paper that the deletion of the 50 bp sequence from KARS101 completely abolished replicator activity in both plasmids and chromosome. These results demonstrate that the sequences required for ARS function on plasmids (Fabiani et al., 1996) are also essential for chromosomal origin function.

KARS101 and pKD1 replicator, but not KARS12, function in both K. lactis and S. cerevisiae. Both KARS101 and pKD1 have a match to the S. cerevisiae ACS present in the 50 bp conserved sequences that is not present in KARS12. We have shown previously that this ACS sequence in KARS101 is required for replicator activity in S. cerevisiae (Fabiani et al., 1996). The results presented in this paper extend this conclusion by showing that the binding of proteins from S. cerevisiae nuclear extracts to KARS101 is dependent on this ACS. In addition to the ACS, sequences important for ORC binding in S. cerevisiae are also present downstream of the ACS in the 50 bp conserved sequence (Lee and Bell, 1997; Bogan et al., 2000). Linker substitution mutations in these sequences abolished ARS activity in S. cerevisiae (Fabiani et al., 1996) and also diminished the ability of KARS101 to bind proteins from the S. cerevisiae nuclear extract. The presence of these sequences explains the ability of these K. lactis replicators to function in S. cerevisiae, and the proximity of the essential sequences for the initiation of replication in both K. lactis and S. cerevisiae may reflect an important role played by adjacent regions promoting replicator activity in both organisms. These sequences may be important for the recruitment of proteins involved in the initiation of DNA replication.

The 50 bp sequences in all three K. lactis replicators are A+T rich, with an identity of 60% between KARS101 and KARS12 and 50% between KARS101 and pKD1. The higher homology is limited, for all of them, to the region important for origin function, and the more conserved nucleotides are the As and Ts. They are characterized not by a specific conserved sequence but by repeats of three or more consecutive adenines or thymines. The presence of these repeats reflects a similarity with S. pombe ARS sequences, in which the presence of A and T stretches is a common feature among the different origins analysed. However, S. pombe replicator structure is more complex. S. pombe replicators appear to have a modular organization. They are much longer than the S. cerevisiae ARS element, a few hundred basepairs, and do not have a consensus sequence essential for replication. Replicator activity in S. pombe seems to depend not on a specific sequence, but on the number and length of A and T stretches (Kim and Huberman, 1998; 1999; Okuno et al., 1999). It has been shown recently that S. pombe ORC interacts with the adenine/thymine stretches (Takahashi and Masukata, 2001; Takahashi et al., 2003) through the N-terminal domain of the SpOrc4 protein (Kong and DePamphilis, 2001; 2002; Lee et al., 2001).

In addition to stretches rich in adenines and thymines, the 50 bp sequence important for KARS101 replicator activity also contains direct and inverted repeats. Inverted repeat sequences are a common feature of prokaryotic and eukaryotic control regions including replication origins. Inverted repeats (IR) have the potential to form cruciforms and stem–loop structures, and these structures can influence chromatin structure. It has been shown previously that histones and/or nucleosomes bind less efficiently to inverted repeats, stem–loop and cruciform DNA (reviewed by Pearson et al., 1996). These structures can have different roles, a direct one by making a nucleosome-free DNA more accessible to DNA-binding proteins, or an indirect one by phasing nucleosomes in a replication-permissive manner. In a recent paper, Novac et al. (2002) showed a direct correlation between cruciform structure and binding of protein involved in the regulation of replication initiation in mammalian cells.

We hypothesized that the 50 bp sequence essential for the replicator activity of KARS101 could represent the binding site of protein/s involved in the initiation of DNA replication. In this paper, we show by gel retardation assay that this sequence is required for efficient binding of proteins in K. lactis nuclear extracts. Using different mutant sequences as competitors, we showed that the sequence previously defined as the K. lactis core is required for efficient binding. These results provide support for the idea that the proteins interacting with the core sequence are involved in replication initiation.

The K. lactis ORC homologue is a good candidate for the binding activity. The K. lactis ORC1 gene was isolated and completely sequenced (Gavin et al., 1995). The protein is 50% identical and 68% similar to S. cerevisiae Orc1p. The region conserved among Orc1p in diverse organisms is within the regions involved in ATP binding and hydrolysis. ORC binding requires ATP in different organisms, and we examined this requirement in the binding properties of KARS101. The results suggest that the binding of K. lactis nuclear proteins to KARS101 is ATP dependent.

The downstream sequences of the KARS101 essential core bind to proteins in vivo (data not shown) and are particularly rich in A+T (about 80%) and, as was suggested for the B2 element in S. cerevisiae ARS307, they may represent a DNA unwinding element (DUE) (Lin and Kowalski, 1997). The AT richness could facilitate the unwinding of the sequence after ORC binding to allow the binding of proteins important for the formation of the prereplicative complex. It was shown recently by Wilmes and Bell (2002) that the S. cerevisiae B2 element in ARS1 not only has helically unstable DNA but also contains specific sequences related to the ACS that are important for protein binding. The most likely candidates are the MCM proteins because their association with an easily unwound region of the origin is in agreement with their role as the putative eukaryotic replicative helicase. In a comparison of the K. lactis core sequence with the downstream sequences involved in protein binding in vivo, we detected the presence of sequences in direct and inverse orientation that are related to the sequences within the core. Together, these observations support our use of the K. lactis replicator KARS101 as a model to understand the role of the sequences close to the essential core, which may be involved in the interaction of proteins not yet discovered and probably important for the regulation of the activity of chromosomal replicators that is currently poorly understood.

Experimental procedures

Strains and media

Kluyveromyces lactis strain MW98-8C (uraA, arg, lys, K+) was used for transformation by electroporation (Becker and Guarente, 1991). Yeast strains were grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose). YPD plates were supplemented with 1.5% Difco agar. Yeast transformants were selected on –Ura plates (0.67% Difco yeast nitrogen base without amino acids, 0.5% casamino acids, 1 M sorbitol, 2% glucose and 1.5% Difco agar).

Escherichia coli strain DH5α was used for amplification of plasmid DNA (Boyer and Roulland-Dussoix, 1969). E. coli transformants were grown in Luria broth supplemented with 100 µg ml−1 ampicillin (Sambrook et al., 1989).

Kluyveromyces lactis centromeric plasmid pLF1

The 527 bp HinfI fragment, containing the essential sequences of K. lactis centromere II (Heus et al., 1993), was cloned in the AatII site of the pRS306 plasmid to make pLF1. Both plasmid and insert were treated with T4 DNA polymerase before ligation. Various K. lactis chromosomal fragments were cloned in the polylinker of pLF1 and assayed for ARS activity in K. lactis (Becker and Guarente, 1991).

Isolation of replicating yeast DNA

Yeast cultures were grown to mid-log phase, mixed with 0.5 volumes of an ice-cold slurry of azide stop solution (0.5 M NaOH, 0.4 M EDTA, 0.2% sodium azide, pH 8.65) (McCarroll and Fangman, 1988) and centrifuged immediately at 4°C. The cells were then washed twice with sterile water. DNA was prepared by the method of Huberman et al. (1987).

Restriction enzyme digestion and two-dimensional gel electrophoresis

DNA (10–20 µg) was digested as described by Greenfeder and Newlon (1992). The DNA was ethanol precipitated and resuspend in 50 µl of TE (10 mM Tris and 1 mM EDTA, pH 8.0). The entire digest was loaded in one well of an agarose gel for two-dimensional analysis. Two-dimensional gels were run as described by Brewer and Fangman (1987).

Southern blot analysis

DNA from two-dimensional gels was blotted to Nytran membranes (Schleicher and Schuell) according to the procedure of Smith and Summers (1980). The filters were hybridized at 65°C in 5× SSC, 50 mM sodium pyrophosphate, 5× Denhardt's solution, 0.5% SDS and 100 µg ml−1 calf thymus DNA. Probes, isolated by restriction enzyme digestion of plasmids or amplified by PCR, were labelled with a Multiprime Random Primer DNA labelling kit (Amersham). Probe was added to DNA filters after prehybridization for 1 h in hybridization solution. Blots were exposed to Kodak XR film with an intensifying screen at −80°C.

Isolation of a KARS101 genomic fragment from a K. lactis genomic library

The K. lactis genomic bank, a generous gift from M. Wesolowski, was prepared by cloning Sau3A restriction fragments from K. lactis chromosomal DNA into the BamHI site of a derivative of the YIp5 plasmid (Sikorski and Hieter, 1989) with the S11 fragment of the pKD1 plasmid (Chen et al., 1986) cloned in the EcoRI site. We amplified the library and screened E. coli transformants by colony hybridization using the 913 bp HindIII–PstI fragment to identify a genomic fragment containing KARS101. Plasmid pCC10 carries a KARS101 genomic fragment of about 6 kb.

Construction of the KARS101 deletion plasmid pLF101

The S. cerevisiae URA3 gene, which complements the K. lactis uraA mutation, was isolated from the YDp-U plasmid (Berben et al., 1991) on a 1.1 kb BamHI fragment. The fragment was blunt ended with T4 polymerase and inserted as a selectable marker in the XhoI site (previously blunt ended with T4 polymerase) of the KARS101 genomic fragment present in the plasmid pCC10. We digested the pCC10 plasmid with SspI, blunt ended with T4 polymerase and, after digestion with XbaI, we purified the 3.1 kb fragment carrying the URA3 gene inserted in the XhoI site. This fragment was cloned in a derivative of pLF1 carrying a 49 bp fragment of KARS101 extending from the ClaI site to position 49 of the 103 bp sequence (see Fig. 1). This KARS101 fragment lacks the essential core sequence. To insert the URA3 fragment, the pLF1 derivative was first digested with BglII, which cuts in the polylinker adjacent to the KARS101 fragment, blunt ended with T4 polymerase and finally digested with XbaI. The derived plasmid (with the complete core deletion, URA3 as a selectable marker and a long downstream flanking sequence) was digested with KpnI (present in the polylinker) and, after treatment with T4 polymerase, with ClaI to get compatible ends useful for cloning the upstream 3.5 kb EcoRI–blunt ClaI fragment derived from KARS101 genomic fragment.

Preparation of nuclear extracts from yeast

Saccharomyces cerevisiae strain AH201 and K. lactis strain MW98-8C were grown in YPD to a density of 3.5 × 107 cells ml−1, and the purification of nuclear extracts was carried out by the method of Schneider et al. (1986).

DNA-binding assays

The DNA probes were labelled with DNA polymerase I, Klenow fragment (New England Biolabs) and then isolated by PAGE. The competitor DNA in the DNA-binding reaction was either the plasmid pRS306 or poly(d(AG))–poly(d(CT)) (Pharmacia). Results obtained with the two competitors were similar. The typical binding reaction mixture contained 25 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 0.8 mM ATP, 70 mM KCl, 2 mg ml−1 BSA, 5 mM dithiothreitol, 5% glycerol, 0.1 mg ml−1 competitor DNA, 0.5 ng of labelled probe (10 000 c.p.m.), corresponding to the 121 bp SalI–BamHI fragment isolated from the centromeric plasmid pLF11, which carries the 103 bp KARS101 ClaI–SspI fragment cloned between the ClaI and SmaI sites of the polylinker, and the indicated amount of nuclear extract (Rao and Stillman, 1995). The protein–DNA binding reaction was incubated for 30 min at 25°C in 20 µl final volume, and then the reaction sample was loaded onto a 5% non-denaturing polyacrylamide gel in 0.25× Tris–borate–EDTA (TBE) (acrylamide/bisacrylamide, 29:1). The gel was run for 3 h at a constant voltage of 200 V at 4°C. The gel was dried and then autoradiographed.

The sequence between nucleotides 50 and 100 of KARS101 was synthesized as complementary oligonucleotides, which were annealed in vitro and used as specific competitor (Fig. 5A) and as probe in the bandshift reactions (data not shown).

The sequences used as competitors in Figs 5B and 6B were prepared using pRS306-derived plasmids as templates for PCR amplification of wild-type and mutant KARS101 sequences, with the primers shown in Fig. 5A. The fragments amplified included the wild-type 103 bp ClaI–SspI KARS101 fragment (pLF11) and deletion and substitution derivatives of this fragment (49-58, 54-63, 68-77, 79-88 and 87-96) (Fabiani et al., 1996) cloned in the ClaI–SmaI sites of the polylinker. The PCR products used as competitors in the binding reactions were purified on 2% agarose gels, and those used as probes were purified on a 7% native polyacrylamide gel (50:1 acrylamide/bisacrylamide in 0.5× TBE) after digestion with SalI and BamHI and fill in with Klenow.

In vitro footprinting

After the binding reaction described above, reaction mixtures were chilled, and different amounts of DNase I (0.1, 0.15, 0.2 and 0.25 units) were added. The incubation was carried out for 1 min at 0°C to get at least one cut per molecule of DNA. The digestion products were separated by electrophoresis on native polyacrylamide gels, as described by Singh et al. (1986). After autoradiography, DNA was eluted overnight at room temperature. The recovered DNA was analysed on 15% denaturing polyacrylamide gels. The DNase footprints were detected by autoradiography.

Electrophoretic profiles of selected lanes were subjected to densitometric scanning, and the value of each band intensity was normalized to the sum of the total intensity of the lane. The data are reported as the ratio, point by point, between profiles from in vitro DNase I-treated samples and DNase I-digested naked DNA.

Acknowledgements

We thank Jim Theis, Ida Ruberti, Paola Ballario, Michele Saliola and the members of the Newlon laboratory group for helpful discussions throughout the study. This work was supported by MURST (Ministero Università Ricerca Scientifica Tecnologica – Progetti Ateneo and Progetti Facoltà), Istituto Pasteur Fondazione Cenci Bolognetti and NIH (grant number GM35679 to C.S.N.). L.F. was supported by a CNR short-term fellowship. C.I. is a PhD student on Cellular Biology, Università‘La Sapienza’, Roma. C.M. is a PhD student on Pasteurian Sciences, Università‘La Sapienza’, Roma.

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