The spatial arrangement of ORC binding modules
determines the functionality of replication
origins in budding yeast
Yung-Tsi Bolon and Anja-Katrin Bielinsky*
Department of Biochemistry, Molecular Biology and Biophysics, 6-155 Jackson Hall, 321 Church Street SE,
Minneapolis, MN 55455, USA
Received June 2, 2006; Revised August 22, 2006; Accepted August 28, 2006
In the quest to define autonomously replicating
sequences (ARSs) in eukaryotic cells, an ARS
consensus sequence (ACS) has emerged for bud-
ding yeast. This ACS is recognized by the replica-
tion initiator, the origin recognition complex (ORC).
However, not every match to the ACS constitutes a
replication origin. Here, we investigated the require-
ments for ORC binding to origins that carry multiple,
redundant ACSs, such as ARS603. Previous studies
raised the possibility that these ACSs function as
individual ORC binding sites. Detailed mutational
analysis of the two ACSs in ARS603 revealed that
they function in concert and give rise to an initiation
pattern compatible with a single bipartite ORC bind-
ing site. Consistent with this notion, deletion of one
base pair between the ACS matches abolished ORC
binding at ARS603. Importantly, loss of ORC binding
in vitro correlated with the loss of ARS activity
in vivo. Our results argue that replication origins in
yeast are in general comprised of bipartite ORC
binding sites that cannot function in random align-
ment but must conform to a configuration that
permits ORC binding. These requirements help to
explain why only a limited number of ACS matches
in the yeast genome qualify as ORC binding sites.
Although the mechanism underlying the initiation of DNA
synthesis differs across species, a general theme has emerged
to describe the activation of the replication process in proka-
ryotes, animal viruses and simple eukaryotes. The recognition
of a binding site with multiple sequence elements is a key
factor for the initiation of DNA replication in a number of
organisms (1). For many of these sites, the spacing between
sequence elements is crucial for the binding of the replication
initiator and the subsequent initiation of DNA synthesis (2). It
has been shown, for instance, that exact spacing of the bipart-
ite initiator binding site is necessary for replication from oriP
in Epstein-Barr virus (3) and the simian virus (SV) 40 origin
(4). The situation in eukaryotic cells is less well defined, as
replication initiates from multiple start sites distributed along
chromosomes and the search for origin sequences, especially
in the genomes of mammalian cells, has proven difficult, with
only a few well-studied examples (5).
Saccharomyces cerevisiae remains the eukaryotic system
in which replication origins are best understood. Origin
sequences were first identified as autonomously replicating
sequences (ARSs) by their ability to promote replication of
plasmid DNA (6–8). ARSs carry multiple matches to an
AT-rich, 11 bp ARS consensus sequence (ACS), 50-(A/T)
TTTA(C/T)(A/G)TTT(A/T)-30(8–10). Substitution mutations
scanning origin regions with matches to the ACS have iden-
tified multiple functional elements (11–15). Elements that are
essential for replication are called A elements, while those
that are non-essential are referred to as B elements (14). In
ARS1, A and B elements overlap with matches to the ACS
(Figure 1A) and at least one B element in conjunction with
the A element is required to form a functional origin (16,17).
Other ARS sequences, including ARS305 and ARS307
(Figure 1B), contain similar clusters of a single, essential A
element and multiple B elements, all of which are associated
with an ACS (13,15,18). What has remained a conundrum is
the fact that there are more than 10 000 matches to the ACS
in the budding yeast genome, but only a small subset of these
appear to be functional binding sites for the initiator, the ori-
gin recognition complex (ORC) (19). In an attempt to distin-
guish functional ACSs from non-functional ones, an extended
ACS that comprises 17 bp, instead of 11, has been described
(20). However, the predictive value of this 17 bp sequence
has not been firmly established, and it seems that algorithms
based on the 17 bp consensus sequence (21) have a limited
capacity for correctly predicting all 430 potential origins in
the yeast genome (19).
Footprinting analyses have shown that ORC interacts dir-
ectly with the ACS (18,22–24). In contrast to origins such
as ARS1 and ARS307, which carry a single essential A
*To whom correspondence should be addressed. Tel: +1 612 624 2469; Fax: +1 612 625 2163; Email: firstname.lastname@example.org
? 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Published online 19 September 2006Nucleic Acids Research, 2006, Vol. 34, No. 185069–5080
element, a second class of origins, branded compound ori-
gins, has been described (25,26). These consist of multiple
ACS matches that appear to serve as redundant ORC binding
sites because the origin was still active when one ACS was
mutated (26). The best-studied examples for this class are
ARS101 (Figure 1C) and ARS310 (26). Similarly, ARS603
is also comprised of two ACSs (Figure 1D) (27). We have
designated the ACS matches in ARS101 and ARS603 as A1
and A2, respectively (Figure 1C and D); however, it is
important to point out that they are not directly equivalent
to the A elements in ARS1 and ARS307, which are necessary
for ORC binding but not sufficient (15–18). Substitution
mutations at either ACS in ARS603 do not abolish origin
activity, but combined mutations in both ACSs result in com-
plete loss of replication function (27). Thus, ARS603 has been
proposed to function as a compound origin with multiple
individual ORC binding sites (26).
One prediction for compound origins is that they possess
multiple, redundant replication start sites (26). Previous map-
ping studies of ARS1 revealed a single, defined leading strand
start site that maps adjacent to the ORC binding site (28,29).
For origins with redundant, non-essential ACSs, it is therefore
possible that more than one start site for replication exists.
However, this has never been directly addressed.
To examine the origin structure of an ARS element con-
taining more than one potential ORC binding site, we invest-
igated replication initiation at the origin ARS603. Our studies
revealed that replication initiates in close proximity to the
ACS matches where ORC binding occurs. The initiation pat-
tern of ARS603 is similar to that of ARS1 (29), suggesting that
the two ACSs at ARS603 do not function as individual
origins. Because it was previously unknown if a single or
multiple ORC complexes occupy the ACSs in compound ori-
gins, we employed electrophoretic mobility shift assays
(EMSAs) to distinguish between these possibilities. Our res-
ults demonstrate that the two ACSs act in concert as a single
bipartite ORC binding site and that their spacing and orienta-
tion affect ORC binding in vitro and in vivo. While substitu-
tion mutations similar to those introduced in earlier studies
(27) retained some ORC binding activity, deletion of one
ACS abrogated ORC binding. These studies argue that a sin-
gle ACS is insufficient to define a functional ORC binding
site, and we propose the inclusion of bipartite consensus
sequences in future algorithms for the identification of replica-
tion origins in yeast. The dependence of ORC binding on the
spacing of specific recognition sequences identifies another
link between origin structure and activation to further our
understanding of the DNA replication initiation process.
MATERIALS AND METHODS
Replication initiation point mapping
Replication initiation point (RIP) mapping was performed on
the strain SP1 MATa, ura3-52 his3 trp1-289 leu2-2,113 ade8
can1 (14). DNA was isolated by CsCl gradient centrifugation
from asynchronously growing cultures, as described (30).
As a source for the non-replicating DNA control, DNA was
isolated from nocodazole-arrested cells. Approximately
2 mg of isolated DNA was digested with PstI, leaving a
50phosphorylated end, and then treated overnight with
l-exonuclease to eliminate nicked DNA (30). The sample
was then used as a template for primer extension reactions
using radiolabeled primers as described (30). Reactions
were fractionated on a polyacrylamide gel next to corres-
ponding sequencing reactions. The sequences for the primers
utilized in this study were as follows: 161t (50-GCTGGG-
AGAACTATTCTTCCAGAG-30) for the top strand and
665b (50-GAGCGATTCTGATGAAACGCACTG-30) for the
In vitro ORC EMSA
Binding reactions were performed with DNA fragments
generated by PCR using biotin-labeled primers. To reduce
variability, one set of biotin-labeled primers (IDT) was
used to recognize the flanking regions of the cloning site in
the plasmid pSTBlue-1 (Novagen) (31). Annealed oligonuc-
leotides spanning wild-type (WT) or mutant origin sequences
of ARS603, ARS101, ARS1 and ARS307 were inserted into
this vector. The length of the probes ranged from 81 to
100 bp to include core sequences for each origin (Figure 1)
and 44 bp of vector flanking regions. Binding reactions
with 10 fM of labeled probe and 10 nM baculovirus-purified
yeast ORC, a generous gift from Dr B. Stillman, were incub-
ated in the presence of 1.5 mM ATP, 1 mM DTT, 0.5 mM
phenylmethlysulfonyl fluoride (PMSF), 20 mM KCl, 5 mM
MgCl2, 1 mM Tris (pH 7.5), 22.5 mM HEPES (pH 7.0),
0.2 mM EDTA, 2 mg/ml BSA, 2.5% glycerol and 25 mg/ml
poly(dG–dC) as nonspecific competitor. These conditions
were used to shift ?50% of the probe for WT ARS603.
Biotin-labeled DNA was visualized using a chemilumines-
cent detection kit (Pierce).
Figure 1. Core ACS sequences for four origins ARS1, ARS307, ARS101
and ARS603. (A and B) The ACSs at ARS1 and ARS307 are shown in boxes.
Previously defined A and B1 elements (14,15,17) are indicated by bold lines.
(C and D) The ACSs at the compound origins ARS101 and ARS603 are shown
in boxes (26,27), and the A1and A2elements are indicated by bold lines.
5070Nucleic Acids Research, 2006, Vol. 34, No. 18
Chromatin immunoprecipitation (ChIP)
Strains used for integration of ARS603 into the URA3 locus
and mutation of endogenous ARS603 by two-step gene
replacement were derivatives of BF264-15DU MATa ura3
ade1 his2 leu2-3,112 trp1-12 Dns (32). For integration at
the URA3 locus, 206 bp of ARS603 was ligated into the
SacI–SalI cloning region of pRS306. The vector was then
linearized with StuI within the URA3 marker and integrated
into the chromosomal URA3 locus. Chromatin samples
were obtained from asynchronously growing cultures and
processed as described (33). ORC-bound fragments were pre-
cipitated from pre-cleared chromatin with a mouse, anti-yeast
ORC2 monoclonal antibody (RDI) and protein A sepharose
beads (Sigma), which were pre-blocked in BSA. Immuno-
precipitated DNA fragments were detected by PCR. Primer
pairs for the ChIP analysis were specific to the following
regions: ARS1: 630 (50-GTTAGCTGGTGGACTGACGCCA-
GAAA -30), 1010 (50-GCCTGTGAACATTCTCTTCAAC-
AAG-30); R11: t(50-CACCGATACGTACTTAAACTCT-30),
2613b (50-CACCTCTGACTTGAGCGTCGAT-30); ARS603: 161t
(50-GCTGGGAGAACTATTCTTCCAGAG-30), 482b (50-CTC-
Quantitation by particle analysis was performed using the
NIH ImageJ program, and fold enrichment was calculated
by taking the ratio of immunoprecipitated to input signals
for the origin region (ARS1 or ARS603) divided by the
same ratio for the non-origin region (R11) as described (34).
Plasmid stability assay
Origin sequences ?200 bp in length were cloned into
pSTBlue-1 (Novagen) unless otherwise indicated. Site-
directed mutagenesis reactions were performed according to
the Stratagene QuikChange procedure. The sequences were
then cloned into the pARS plasmid vector containing the
URA3 gene and a centromere (14). Yeast transformations
into the SP1 strain (14) were performed with lithium acetate,
and cells were plated onto SC–Ura plates. Primary transform-
ants were further streaked out on an additional SC–Ura plate.
Yeast cells containing the pARS plasmid with the given
origin sequence were grown in SC–Ura medium to early
log phase, counted by hemocytometer and plated to assess
the initial number of plasmid-containing cells. Approximately
200 cells were released into 2 ml of complete medium for
30 h at 30?C. Cells were counted again, and equal amounts
were plated on complete and SC–Ura medium to determine
the percent of colonies that retained the pARS plasmid. The
assay was duplicated for at least two independently isolated
transformants of each strain.
Replication in ARS603 initiates close to the
two ACS elements
Previously, we investigated DNA replication events at ARS1
and found that DNA synthesis initiates adjacent to the site of
ORC binding (28,29). To determine the DNA replication
initiation pattern for ARS603, we performed RIP mapping.
This method allowed us to map the start sites for both leading
and lagging strand synthesis with nucleotide resolution (30).
ARS603 has two core elements, a 10/11 and a 9/11 match to
the ACS (27). Here we refer to the first ACS match as A1and
the second ACS match as A2(Figure 1D). A1and A2are ori-
ented in opposite directions, separated by 5 bp. For RIP map-
ping, DNA was isolated by CsCl gradient centrifugation from
asynchronously growing cultures. Nocodazole-arrested cells
served as the source for the non-replicating DNA control
sample (30). Nascent DNA was enriched by treatment with
l-exonuclease (28,30). Nascent or non-replicating DNA
was used as templates for primer extension reactions using
radiolabeled primers that annealed ?250 bp up or down-
stream of the ACSs. Primer extension products were frac-
tionated on polyacrylamide gels next to corresponding
sequencing reactions (30). The ladder of bands represents
replication intermediates (Figure 2A and B) and the position
of the smallest band marks the initiation site for the leading
strand [also referred to as the transition point (TP) between
leading and lagging strands, TP; Figure 2A and B]. All other
bands represent initiation sites for Okazaki fragments (29).
Any faint bands that were not reproducible were not con-
sidered. We mapped the TP between leading and lagging
strand synthesis within the A2element (Figure 2A and B).
Replication initiated at nucleotide position 403 on the top
strand and at nucleotide position 394 on the bottom strand,
9 bp apart. Subsequent Okazaki fragment startsites for lagging
strand synthesis were spaced at intervals of ?50–60 bp or
multiples thereof. DNA from nocadozole-arrested cells did
not show any reproducible bands, as expected (Figure 2A
and B, lane N). Positive controls for correct primer anneal-
ing were performed on restriction enzyme-digested DNA,
where the restriction site was confirmed by RIP mapping
(data not shown). A synopsis of the initiation map of
ARS603 is shown (Figure 2C). It is worthwhile to note
that we did not observe a composite pattern (an overlay of
two initiation maps with one replication start site in A1and
another one in A2), as might have been predicted for a com-
pound origin (Figure 2D, bottom; Figure 2E, lane D). These
results suggest that DNA replication at ARS603 likely initi-
ates from a single start site, similarly to ARS1 (28,29).
Each ACS match at ARS603 contributes equally
to ORC binding
Based on our RIP mapping results and earlier studies on
ARS603 by others (27), we predicted that ORC binds to A1
and A2. Because this had never been tested directly, and it
was unclear whether A1and A2could bind a single ORC or
multiple complexes, we performed electrophoretic mobility
shift analyses on ARS603 using purified ORC. Initially, we
titrated ORC to approximate binding of 50% of a ?90 bp
probe (data not shown) and used these conditions for
subsequent experiments (see Materials and Methods for
details). ORC binding to ARS603 was dependent on the pres-
ence of ATP (data not shown), as expected (24,35,36).
Previous analyses of ARS603 used XhoI substitution
mutations that converted the A1and A2sequences into 7/11
matches to the ACS. Either mutation at A1or A2alone did
not affect origin activity; only the combined mutation of both
elements resulted in complete loss of activity at ARS603 (27).
Nucleic Acids Research, 2006, Vol. 34, No. 185071
Using these exact substitutions, here labeled S4, we found
that mutations at A1or A2caused a decrease in ORC binding
in comparison to ORC binding at the WT ARS603 sequence
(Figure 3A). Moreover, the combined mutation of both A1
and A2resulted in the dramatic reduction of ORC binding,
consistent with the reported loss of origin activity (27).
These results suggested that ORC binding is indeed affected
by mutations in A1or A2.
Since the XhoI substitution mutations resulted in a gain of
GC content, we tested a variety of base substitutions that
deviated from the ACS, but kept the GC content of A1and
A2the same. Not surprisingly, the increase in mismatches
to the ACS correlated with the decrease in the amount of shif-
ted probe (Figure 3B), as expected from prior findings (24).
Interestingly, however, these 7/11 matches retained much
less ORC binding capacity than the 7/11 matches in the S4
Figure 2. Initiation pattern of ARS603. (A) RIP mapping on the top strand of ARS603 shows the transition point (TP) between leading and lagging strand
synthesis at the A2element. Replication intermediates (RIs) are fractionated on a polyacrylamide gel next to corresponding Sanger sequencing reactions (C,G).
Okazaki fragment initiation sites are indicated above the TP by black dots. Arrows denote the position and orientation of the A1and A2elements. As a negative
control, DNA from nocodazole-arrested cells (N) was used as a template. (B) RIP mapping on the bottom strand of ARS603 reveals the transition point (TP) at the
A2element, and the Okazaki fragment initiation sites are also marked by black dots. Replication intermediates (RIs) are shown next to corresponding Sanger
sequencing reactions (G,T). DNA from nocodazole-arrested cells (N) was used as a negative control. (C) A synopsis of the initiation sites at ARS603 is shown.
Bold arrows indicate the transition points (TPs) on the top and bottom strands, and the other arrows indicate lagging strand initiation sites. Nucleotide positions of
individual start sites are shown. (D) Initiation maps for ARS603 with a single TP (bold arrows, top) or hypothetical dual TPs (bold arrows, bottom) if the origin
initiates replication at either position with similar frequencies. For dual initiation sites, two closely spaced (?10 nt) TPs in A1and A2are shown, as predicted for
a compound origin with two independent ORC binding sites. Lagging strand initiation sites also lie ?10 nt apart, a distance too short to accommodate
consecutive Okazaki fragments. (E) A cartoon of hypothetical RIP mapping results shows a polyacrylamide gel with either single (S) or dual (D) initiation sites
on the top strand of ARS603. Sequencing reactions (C,G) identify the position of the ACSs indicated by arrows (A1, A2), as in (A). The location of the TP for the
single initiation site scenario (TPS) is indicated by a horizontal arrow, and the location of the TPs for the dual initiation site scenario (TPD) is bracketed to
encompass both arrows.
5072Nucleic Acids Research, 2006, Vol. 34, No. 18
A2?and S4 A1?probes (compare Figure 3A and B). The
decrease in binding activity may have been a result of mutat-
ing the highly conserved position 10 (8) within the ACS of
A1?and A2b?probes. In contrast, the S4 mutations retain a
stretch of Ts in positions 8–10, leaving the most highly con-
served nucleotides intact (8). To further disrupt each A ele-
ment in ARS603, we utilized substitution mutations with the
linker sequence (50-CCTCGAGG-30). These linker sequences
replaced 8 bp of sequence and were used in earlier studies to
dissect ARS1, ARS307 and ARS305 (13–15,18). We called
these mutations S6 because they replaced 6 bp of the ACS,
reducing the match at these elements to 5/11 (Figure 3C).
The ability of ORC to retard the electrophoretic mobility of
the ARS603 fragment was severely decreased for both S6
fragments (Figure 3C, lanes A1?and A2?). In contrast to
the S4 probes, ORC binding was reduced by >50% for indi-
vidual S6 probes [similar to the A1?and A2b?fragments
(Figure 3B)]. Equivalent mutations in A1and A2resulted
in similar reduction of ORC binding, suggesting that the indi-
vidual ACSs play an equal role in ORC binding at ARS603.
These results suggested to us that A1and A2contribute syner-
gistically to ORC binding, either as two individual but cooper-
ative ORC binding sites or as a single ORC binding site.
A single ACS match is insufficient to support
Although both ACSs in ARS603 appeared to contribute
equally to ORC binding, we never detected a second band,
Figure 3. A1and A2contribute equally to ORC binding at ARS603. EMSAs were performed in the presence and absence of purified ORC on probes containing
the core ARS603 sequence (Figure 1D) flanked by common vector sequences (see Materials and Methods). (A) Mutations that substituted 4 bp in the ACS
reduced ORC binding equally at A1and A2. XhoI substitution mutations (S4) were introduced in single elements (A1?, A2?) or in combination (A1A2?).
Mismatches to the ACS are in gray and underlined. (B) ORC binding ability correlated with the number of matches to the ACS. Mutations with conservation of
GC content still affected ORC binding. Notations below the figure indicate the number of matches to the 11 bp ACS. (C) Mutations that replaced 6 bp of the ACS
reduced ORC binding equally at A1and A2. Substitution mutations with the sequence CCTCGAGG (S6) were introduced at single elements (A1?, A2?) or in
Nucleic Acids Research, 2006, Vol. 34, No. 185073
even at very high concentrations of ORC (50 nM/reaction,
data not shown), as would have been expected if two ORCs
could bind to the WT probe. To further define the roles of A1
and A2, we constructed fragments of the same length as the
WT ARS603 fragment, containing either element A1or ele-
ment A2flanked by random sequences that were unable to
bind ORC by themselves (data not shown). Subsequent
in vitro binding assays revealed no significant binding of
ORC to either A1or A2alone (Figure 4A). The consequences
of deleting A1or A2contrasted with the effect of substitution
mutations that still allowed for ORC binding as long as one
ACS remained intact (Figure 3), explaining earlier reports
on the origin activity of ARS603 mutants (27). In addition,
we used unlabeled A1or A2containing fragments as well
as a WT control in a competition assay measuring ORC bind-
ing to a labeled WT ARS603 fragment. Consistent with the
results shown in Figure 4A, 50- and 500-fold excess of
A1or A2sequences did not impede ORC binding to WT
ARS603 (Figure 4B). These data support the notion that
both A1and A2are required for ORC binding to ARS603
and cannot function as individual ORC binding sites.
ORC binding at ARS603 requires a defined
Examination of the ARS603 sequence revealed that A1and A2
are separated by 5 bp. Because A1and A2lie on opposite
strands, this particular spacing would place the two elements
on the same side of the DNA double helix. To explore
whether this specific configuration is a requirement for
ORC binding, we inserted sequences of different lengths
between the A1 and A2 elements. The insertion of 5 bp,
approximately half a helical turn, caused a severe reduction
in ORC binding (Figure 4C). However, insertion of an addi-
tional 5 bp for a total of 10 bp, or close to a full helical turn,
partially restored ORC binding (Figure 4C). Based on these
results, A1 and A2 require alignment to a defined helical
phase to function as an efficient ORC recognition sequence.
A single base pair deletion between the ACS matches in
ARS603 and ARS101 abolishes ORC binding
Replication initiation at a number of viral origins is severely
influenced by the spacing between recognition sites (3,4).
Because the configuration of functional elements and associ-
ated ACSs varies from one origin to another in budding yeast
(37), it is widely assumed that the distance between ACSs is
not relevant for origin function. To test this hypothesis
directly, we examined the effect of a single bp deletion
between A1and A2. Surprisingly, ORC no longer bound to
the ARS603 fragment (Figure 5A). The effect of the single
bp deletion, which removed an A-T bp, was attributed to
change in spacing rather than the requirement for that particu-
lar bp or a change in G-C content, because the substitution of
the same A-T bp with a C-G bp produced no discernable
change in ORC binding (data not shown). Thus, the severity
in the loss of ORC binding upon deletion of 1 bp contrasted
strongly with the number of substitution mutations within the
ACS matches required to achieve the same effect (Figure 3).
To determine whether the effect of a single bp deletion was
specific to ARS603 or could be generalized to other origins,
we chose ARS101 to perform a similar experiment. This ori-
gin also possesses two ACS matches, one of which (A2in
Figure 1C) has been reported to support replication individu-
ally (26). A single bp deletion between the two ACSs, origin-
ally 8 bp apart, severely compromised ORC binding at
ARS101, even though we added twice as much ORC to
these binding reactions as we used for our experiments with
ARS603 fragments (Figure 5B). We concluded that the spa-
cing between the A elements in ARS603 and ARS101 is cru-
cial for ORC binding.
Spacing of bipartite elements at ARS1 and ARS307 does
not affect ORC binding
The ACS matches in both ARS603 and ARS101 were reported
to be redundant with respect to their ability to bind ORC or
confer ARS activity (26,27). In contrast to these compound
Figure 4. Both A1and A2are required for ORC binding to ARS603. EMSAs
were performed in the presence and absence of purified ORC. (A) A single
match to the ACS (A1or A2) did not support ORC binding. (B) 50- and 500-
fold unlabeled wild-type ARS603 competed for ORC binding, but single A
element fragments were unable to compete against wild-type ARS603. (C)
Insertions between the A elements were constructed and tested for ORC
binding. While half of a helical turn (ins5) disrupted ORC binding, a full
helical turn (ins10) restored some binding. Percentages below the figure
indicate the amount of probe shifted by ORC (with wild-type levels set to
100%), as quantified by particle analysis (ImageJ).
5074Nucleic Acids Research, 2006, Vol. 34, No. 18
origins, ARS1 and ARS307 contain only a single essential
ACS match (the A element). Previous studies showed that
the A element is required but not sufficient for ORC binding
and that ORC interacts with both the A and B1 elements in
ARS1 (18,24,35). To address whether the spacing between
A and B1 is crucial for ORC binding at ARS1 and ARS307,
we introduced deletion mutations between these elements.
These deletions also reduced the spacing between the
associated ACSs. ORC binding at both ARS1 and ARS307
was not significantly affected by the removal of a single bp
(Figure 5C and E). Furthermore, no effect was seen at
ARS1 regardless of the removal of either a C-G or a T-A
bp (Figure 5C). Indeed, the deletion of even two out of
the 4 bp between the ACSs at ARS1 was not sufficient to sig-
nificantly impair ORC binding (Figure 5D). In ARS307, the
distance between the two ACSs is rather large, spanning 26
bp (Figure 1B). Therefore, we also tested whether the
removal of 5 bp had any effect on ORC binding. However,
we did not detect any changes (data not shown). Therefore,
at ARS1 and ARS307, small changes in spacing between the
ACSs (and thus between the A and B1 elements) do not
appear to be critical for ORC binding, supporting prior
ARS assay results on ARS1 mutants (38). This is also consist-
ent with earlier reports suggesting that the A element at ARS1
can cooperate with any of the non-essential B elements (16).
Orientation of the A elements affects ORC binding at
ARS603, ARS101 and ARS1
Early comparisons of ARS element configurations revealed
that the orientation of ACSs differs from origin to origin
(6). This prompted us to investigate whether ORC binding
was sensitive to changes in the orientation of individual
ACSs at ARS603, ARS101 and ARS1 (Figure 1). Previous
analyses have noted a favored ACS orientation in some
instances for the support of plasmid replication (6,38,39).
Figure 5. A single bp deletion eliminates ORC binding at ARS603 and ARS101, but not at ARS1 and ARS307. EMSAs were performed in the presence and
absence of purified ORC. (A) ORC binding was abolished by a single bp deletion between the ACSs at ARS603. A single bp deletion (del1) was introduced
between the A elements converting GATAC to GATC. (B) A single bp deletion between the ACSs at ARS101 also severely decreased ORC binding. The 8 bp
spacing was reduced to 7 bp (TTATGTTT to TTAGTTT) between the ACSs at ARS101 (del1). (C) Single bp deletions between ACSs associated with A and B1
elements of ARS1, where the original spacing was 4 bp, did not show a significant decrease in ORC binding ability. Two different single bp deletions were made
with either a C-G bp (delC) or a T-A bp (delT). (D) Deletion of 2 bp (del2) between the ACSs at ARS1 also did not significantly affect ORC binding. (E) Deletion
of a single bp (del1) between ACSs associated with the A and B1 elements at ARS307 did not alter ORC binding.
Nucleic Acids Research, 2006, Vol. 34, No. 18 5075
At ARS603, the reversal of either A1or A2resulted in a
significant reduction of ORC binding (>50%, Figure 6A).
The reversal of either ACS match at ARS101 resulted in a
complete loss of ORC binding (Figure 6B). Meanwhile, at
ARS1, the reversal of the ACS within the essential A element
caused a severe reduction in ORC binding (Figure 6C, lane
revA), similar to a linker substitution mutation in the A
element (Figure 6C, lane link A?). However, a change in
the orientation of either the ACS that is associated with the
B1 element (Figure 6C, lane revB) or the reversal of the
entire B1 element (data not shown) reduced ORC binding
only slightly. According to these results, ORC binding is
sensitive to the orientation of the ACS within the respective
A elements of all tested origins. This further supports the
notion that a single ACS cannot function as an ORC binding
site but acts in concert with its flanking region.
ORC binding in vivo reflects ORC binding in vitro
Because the in vitro DNA mobility shift assays using purified
ORC revealed that the spacing between A1and A2was crit-
ical for ORC binding at ARS603, we next examined the effect
of the single bp deletion in vivo by ChIP using ORC2-specific
antibodies. Regardless of whether ARS603 was inserted at the
URA3 locus or present at its endogenous locus, a single bp
deletion between the A elements abrogated ORC binding in
vivo (Figure 7A and C). In contrast, ORC binding to ARS1
remained unaffected (Figure 7B and D). The non-origin
region R11 was included as a negative control (Figure 7B
and D). Thus, the ability of ORC to bind ARS603 in vitro
accurately reflected its behavior in vivo.
Origin activation parallels ORC binding
So far, we have shown that substitutions within the ACS as
well as changes in orientation and spacing between A1and
A2 severely affected ORC binding at ARS603. To test
whether these mutations also affected origin activation, we
performed plasmid stability assays. These assays require ori-
gin sequences to be cloned into plasmids containing the
URA3 gene and a centromere sequence (40). Once trans-
formed into yeast, the plasmid constructs are stably retained
only with the presence of an active and effective yeast origin.
Mitotic stability indicates the ability of the origin to support
DNA replication initiation and retain the plasmid, thus allow-
ing for survival in selective medium.
Sequences of ?200 bp covering the entire ARS603 region
were used to test origin activity. WT ARS1 served as a posit-
ive control, and ARS1/A?served as a negative control. The
pARS1/A?construct did not produce viable transformants
that could be restreaked onto selective medium, as reported
earlier (14). The level of origin activity in vivo correlated well
with ORC binding in vitro in all cases examined (Figure 8A).
While WT ARS603 had a mitotic stability similar to that of
ARS1, the double substitution mutants for A1 and A2 at
were unableto grow
(Figure 8A and B). Notably, the deletion of a single base
pair or the insertion of a half helical turn also produced
only microcolony transformants that were unable to be pas-
saged onto selective medium (Figure 8A and B). We obtained
similar results when we deleted a single base pair between the
two ACSs in ARS101 (data not shown). Consistent with our
in vitro ORC binding studies, the insertion of a full helical
turn between the A elements partially rescued growth
(Figure 8B). The 38 bp core region of ARS603 containing
WT A1and A2, which was capable of supporting ORC bind-
ing (Figure 3, WT) also produced transformants that could be
restreaked onto selective medium (Figure 8C, A1+A2). At the
same time, fragments of the same length containing only
either A1or A2of ARS603 were unable to grow on selective
medium (Figure 8C), as expected from their inability to bind
ORC in vitro (Figure 4A and B). Thus, in all cases examined,
loss of ORC binding corresponded well with loss of origin
Figure 6. Reversal of the A element affects ORC binding at ARS603, ARS101
and ARS1. EMSAs were performed in the presence and absence of purified
ORC. (A) Changes in the orientation of A1or A2signficantly decreased ORC
binding to ARS603. Mutations were introduced to reverse the A1element
(revA1) or the A2element (revA2) in ARS603. (B) Changes in the orientation
of A1or A2in ARS101 abrogated ORC binding. ACS orientations were
converted to face inward (inori), or leftward (leftori). (C) ORC binding was
sensitive to the reversal of the ACS within the A element of ARS1. Mutations
created a reversal of the ACS associated with the A element (revA), or the
ACS associated with the B1 element (revB). The negative control (linkA?)
for ORC binding was the linker substitution mutation at positions (858–865)
of element A in ARS1 (18).
5076Nucleic Acids Research, 2006, Vol. 34, No. 18
Analysis of ARS603 provides a new perspective on the con-
tribution of both DNA sequence and structure to origin func-
tion. Because no two origins of DNA replication are identical,
it has been difficult to understand what distinguishes func-
tional from non-functional ACSs. Examination of the struc-
tural framework necessary for ORC binding in budding
yeast provides clues to the complex nature of its interaction
with DNA. In this study, we demonstrate that while two
ACSs at ARS603 contribute equally to ORC binding, they
cannot function independently. The ACSs at ARS603 require
a restricted alignment to form a functional ORC binding site,
and it is reasonable to assume that this may apply to other loci
besides ARS603 in the yeast genome.
Two classes of origins in budding yeast
In the course of defining the parameters that allow for the
efficient binding of ORC to ARS603, we discovered that the
interdomain spacing between A1and A2is critically import-
ant. This phenomenon was not found to be unique to ARS603
since binding of ORC to ARS101 was also responsive to sin-
gle bp interdomain deletions. At the same time, not all of the
origins we tested were affected by such changes, as ARS1 and
ARS307 did not show any significant loss of ORC binding.
Therefore, we propose that two classes of origins exist in
yeast: those that require exact interdomain spacing and those
that are tolerant of spacing manipulation. These differences
directly reflect two different modes of ORC binding. In
ARS603, the two ACSs that constitute the bipartite ORC
binding site appear to contribute equally to ORC binding,
whereas the A element in ARS1 appears to be the predom-
inant site of ORC interaction (41). Furthermore, it is pos-
sible that other ORC-interacting proteins modulate the
affinity of ORC for certain sites in the genome. Indeed, pre-
vious studies have found that another replication initiation
protein, Cdc6, regulates the sequence-specific binding of
ORC at ARS1 (42,43). It will be interesting to see whether
Cdc6-dependent regulation is characteristic for this class of
origins or also applies to ARS603 and ARS101.
ORC binding at bipartite sites
Previous studies suggested that ORC could bind to a bipartite
site comprised of A and B1 elements (ARS1 and 307)
(14,15,18,26). However, prior evaluations of origin function
also described the discovery of origins with multiple ACSs
with redundant function (26). These origins were named com-
pound if they contained redundant ACSs that were proposed
to function as individual ORC binding sites (26). Our study
on ARS603 reconciles these two origin models.
Although ARS603 contains two ACSs (A1and A2), we con-
cluded from our results that these two elements are not acting
as binding sites for separate ORCs but rather as a bipartite
binding site for a single initiator complex. This may also
apply to ARS101 because the deletion of a single bp
Figure 7. A single bp deletion inhibits ORC binding to chromosomal ARS603. ARS603 carrying a single bp deletion (del1) between A1and A2was inserted at the
locations. (A and C) ChIPs were performed using ORC2-specific antibodies. PCR reactions contained ARS1-, ARS603-, ARS603/URA3- or R11-specific primers
and immunoprecipitated (IP) or input DNA (A and C). (B and D) Quantification of PCR fragments was performed using ImageJ (NIH) as described (34).
Nucleic Acids Research, 2006, Vol. 34, No. 185077
between the ACSs, as well as changes in their orientation,
abolished ORC binding in vitro. In addition, ARS101 activity
was markedly reduced when one ACS was mutated, arguing
that ORC strongly prefers to bind to both ACSs (26). At first
glance, our results seem to contradict the finding by Theis and
Newlon that the 10/11 match in ARS101 can retain ARS
activity by itself (26). However, further inspection of the
ARS101 sequence reveals two additional 8/11 matches down-
stream of the 10/11 ACS element (data not shown). It is con-
ceivable that one of these 8/11 matches cooperated with the
10/11 match to form a bipartite ORC binding site. Consistent
with this notion, we show that a single A element in ARS603,
another compound origin, cannot sustain ORC binding. These
data are in agreement with early reports that the 11 bp ACS is
necessary but not sufficient for ARS activity (44). This is fur-
ther supported by the fact that substitution mutations at
ARS101 showed the gain of new hypersensitive sites with the
loss of one ACS element (26), indicating that ORC contacted
a second site in close proximity to the remaining A element.
At ARS1 as well, footprinting studies showed that mutation
of the A element resulted in protection over a 9/11 ACS at
an additional B element (24,35), consistent with the recovery
of a bipartite site. Indeed, in vitro studies determined that two
ORCs cannot bind to two inverted ACSs at ARS1 simultan-
eously (24,45), and other studies have determined that the
stoichiometry of ORC to the ARS1 origin is 1:1 (46). It fol-
lows that the recognition of a bipartite binding site with
two ACSs by a single ORC likely applies to the vast majority
of origins in the yeast genome.
In addition, fluorescence anisotropy studies showed that
ORC is capable of binding to ACS matches associated with
various elements of ARS1 (47); however, only with the pres-
ence of the A element was ORC binding dependent on ATP, a
key factor in the sequence-specific recognition of ORC at ori-
gins (24,35,36). Therefore, it seems likely that certain ORC
conformations dictated by the spatial arrangements of ACSs
may either allow or restrict ATP-hydrolysis by ORC (48).
It is conceivable that the single bp deletion between A1and
Figure 8. ORC binding in vitro mimics ARS activity in vivo. (A) The mitotic stability of different ARS603 constructs was tested. Transformants that yielded
viable colonies surviving passage onto selective medium were designated with a plus sign (+). ORC binding was designated as (+++) for wild-type ARS603 and
ARS1 and (++) or (+) for decreasing levels of binding. Lack of ORC binding was indicated by a minus sign (?). The error bars indicate the standard deviation for
the ARS assay on two separate transformants performed in duplicate. (B) Plasmids carrying wild-type (WT), single bp deletion (del1), and 5 or 10 bp
(ins5, ins10) insertions within the 206 bp of ARS603 were streaked onto selective medium (SC–Ura) to assess growth. (C) The same experiment as described in
(B) was performed with the following constructs: 38 bp wild-type ARS603 core region (A1+A2), element A1alone (A1) and element A2alone (A2).
5078Nucleic Acids Research, 2006, Vol. 34, No. 18
A2in ARS603, or the reversal of A elements in ARS1, ARS101
and ARS603, induced an ORC conformation that failed to
repress ATP-hydrolysis by ORC and did not facilitate stable
binding to DNA (36,49).
As a heteromultimer protein complex (24), ORC subunits
possess the potential to make contact at an origin in a variety
of ways. Modification-interference and missing-contact assays
at ARS1 and ARS305 provided the first look at the diverse nat-
ure of the ORC-origin interaction (41). While ORC contacts in
ARS305 were discerned within a ?50 bp region, ORC cover-
age at ARS1 extended to a ?92 bp range (41). Footprinting
and mobility shift studies also support the ability of DNA
sequences to wrap around ORC, enabling it to contact multi-
ple regions at wide ranges (14,18). Thus, it is possible for ORC
to make contact with sequences over a long distance. How-
ever, ORC may possess limited adaptability, resulting in the
selection of certain bipartite sites and providing one explana-
tion for why the yeast genome harbors only ?430 ORC
binding sites, but has more than 10000 ACS matches (19).
In a recent model for the prediction of functional origins in
budding yeast, Breier et al. devised an Oriscan algorithm
based on the alignment of a subset of known origins to the
extended 17 bp ACS (20,21). This algorithm was based on
the assumption that a single extended ACS (20) in conjunc-
tion with certain flanking regions could function as an ORC
binding site and did not fully take into account the bipartite
structure of replication origins in yeast (18). We propose a
revised algorithm definition to include two matches to the
ACS as a bipartite binding site for ORC, as previous studies
have also shown that synthetic constructs with two copies of
the ACS are sufficient for ARS activity (6). Moreover, it is
known that the presence of additional ACSs further improve
origin function (6,45). This definition does not preclude the
use of the extended 17 bp ACS (20).
Prior analyses of the region between the A and B1 ele-
ments (and thus between the corresponding ACSs) of ARS1
indicated that an insertion of 195 bp or greater essentially
inactivated ARS activity (38). Inspection of nine origins on
chromosome VI with identified, essential ACSs (27,50)
revealed that a second ACS (with a 9/11 match or better)
resides in close proximity, at a distance of at most 119 bp
(data not shown). On average, we calculated the distance to
be 38 ± 35 bp (data not shown). We suggest that these con-
siderations might provide a framework for the definition of a
functional ORC binding site. Clearly, further studies are
needed to test the validity of this hypothesis. Phylogenetic
comparisons of conserved sequences may contribute to
these endeavors (51).
Bipartite structures for origin recognition in
Our studies demonstrate that the spacing of the bipartite ORC
binding site affects the origin activity of ARS603. Replication
mechanisms in a number of prokaryotes and viruses show
similarity to mechanisms employed at the eukaryotic origins
of budding yeast (1). In fact, the recognition of a bipartite
binding site for the initiation of bi-directional replication is
a familiar theme that is seen in bacterial and viral systems,
such as at oriP of Epstein-Barr virus and the core origin of
relies on the presence of two out of the multiple 9-mers that
comprise oriC, consistent with the recognition of a bipartite
site (52). Precise spacing of sequences at the dyad symmetry
element DS within oriP is required for interaction with
EBNA1 in Epstein-Barr virus where origin activity is abol-
ished with the deletion of 1 to 2 bp (3). Likewise, spacing
affects binding of the SV40 initiator, large T-antigen, at site
II that requires a minimum of 10 bp between two pairs of
pentanucleotides with inward orientation (4). According to
our results, the A elements at ARS603 appear to function
similarly with a requirement for defined spacing and helical
Some evidence exists to suggest the conservation of a
bipartite site in other eukaryotes as well. For instance, foot-
printing analyses of a metazoan replication origin, ori II/
9A, in Sciara coprophila using nuclear extract and purified
ORC, show two separate regions of protection for ORC bind-
ing (53). Furthermore, recent ChIP studies at the human
c-myc replicator suggest that ORC binds to two distinct
AT-rich regions, resulting in a bipartite binding pattern
(54). Thus, the use of a bipartite binding site for the initiator
appears to be another connection in the mechanism for the
initiation of DNA replication in multiple organisms.
The authors are grateful to B. Stillman for baculovirus-
purified yeast ORC, the SP1 strain and the pARS plasmid.
The authors would also like to thank S. P. Bell for
D. Clarke for the BF264-15DU strain. In addition, The
authors thank D. M. Livingston, J. Theis and the Bielinsky
labmembersfor advice and
E. A. Hendrickson for critical reading of the manuscript.
This work was supported by ACS grant RSG0216601 to
AKB. Funding to pay the Open Access publication charges
for this article was provided by the American Cancer Society.
ORCand protocols, and
protocols, aswell as
Conflict of interest statement. None declared.
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