Biochimica et Biophysica Acta 1677 (2004) 142 – 157
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Review
The activities of eukaryotic replication origins in chromatin
Michael Weinreich a,*, Madeleine A. Palacios DeBeer b, Catherine A. Fox b,*
a
b
Laboratory of Chromosome Replication, Van Andel Research Institute, 333 Bostwick Ave. NE, Grand Rapids, MI 49503, USA
Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706-1532, USA
Received 31 July 2003; accepted 17 November 2003
Abstract
DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles
of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of
hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For
example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent
studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can
control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient
replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in
chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin
structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit
replication proteins with the potential to form context-dependent protein – protein interactions.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Chromosome; Replication; Chromatin; Origin; Yeast
1. Eukaryotic replication origins: overview and
background
The goal of this review is to summarize selected experiments that provide evidence for roles of chromosome
context and chromatin structure in regulating the activity
of eukaryotic replication origins. The DNA sequences
required for replication origin activity have been extensively defined and characterized in only one eukaryotic organism, the budding yeast Saccharomyces cerevisiae. In
addition, two recent independent and complementary
whole-genome studies of DNA replication in vivo have
identified virtually every potential and active chromosomal
replication origin in this organism [1,2]. For these reasons,
and the tractability of S. cerevisiae as a genetic and
biochemical model for DNA replication, many of the
mechanistic ideas we have about chromatin structure and
its impact on the activity of replication origins have come
from studies in budding yeast. Therefore, this review will
focus primarily on experimental observations made in S.
cerevisiae, but where appropriate, such observations will be
related to other organisms.
This section provides a background on yeast replication
origin activity and organization (Sections 1.1 and 1.2) and
the steps in replication initiation that could be affected by
chromatin structure (Section 1.3). Section 1.4 considers the
potential impact of origin activity and the organization and
regulation of origins within the genome on both chromosome replication and other chromosomal functions.
1.1. Replication of the eukaryotic genome uses many
individual replication origins
* Corresponding authors. M. Weinreich is to be contacted at Laboratory
of Chromosome Replication, Van Andel Research Institute, 333 Bostwick
Ave. NE, Grand Rapids, MI 49503, USA. Fax: +1-616-234-5306. C.A.
Fox, Department of Biomolecular Chemistry, University of Wisconsin
Medical School, 1300 University Ave., Madison, WI 53706-1532, USA.
Fax: +1-608-2625253.
E-mail addresses: [email protected] (M. Weinreich),
[email protected] (C.A. Fox).
0167-4781/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbaexp.2003.11.015
Extensive mechanistic information is available concerning the steps of DNA replication in the model prokaryote E. coli, and many studies indicate that several aspects of
E. coli DNA replication serve as paradigms for eukaryotic
DNA replication. For example, the replicon model for DNA
replication postulated the existence of ‘‘initiator proteins’’
M. Weinreich et al. / Biochimica et Biophysica Acta 1677 (2004) 142–157
that interacted with ‘‘replicator’’ DNA elements to direct
duplication of the prokaryotic genome. Compelling support
for this model came from elegant studies of the DNA
sequences and proteins that controlled the initiation of
DNA replication of the E. coli genome [3]. These classic
studies provided powerful guides for identifying the DNA
sequence elements and proteins critical to replication initiation in eukaryotes [4,5]. It is now well established that both
prokaryotic and eukaryotic cells depend on specific DNA
binding proteins to mark particular genomic regions as sites
to initiate DNA replication.
However, significant differences in both the mechanisms
and regulation of genome replication in prokaryotes and
eukaryotes exist, and one of the most obvious is the number
of replication origins that are used to duplicate the genetic
material. E. coli uses a single replication origin to duplicate
its entire 4.6-Mb genome. In contrast, eukaryotic cells
initiate DNA synthesis from hundreds to thousands of
replication origins distributed over the multiple chromosomes that constitute the genome. For example, in budding
yeast, chromosome III (0.32 Mb) uses 11 origins for its
duplication while chromosome X (0.75 Mb) uses about 20
[1,2,6]. Therefore, multiple replication origins per chromosome is a fundamental and defining feature of the eukaryotic
genome (Fig. 1).
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1.2. Individual eukaryotic origins are functionally distinct
The S. cerevisiae genome contains approximately 400
replication origins, and each individual origin is distinct in
terms of efficiency and activation time during the DNA
synthesis phase (S-phase) of the cell cycle. Replication
origin efficiency refers to the frequency at which an origin
initiates DNA replication (fires) within a population of cells.
In yeast, many origins are efficient, firing in virtually every
dividing cell within a population. However, some replication origins are less efficient, initiating in fewer than half the
dividing cells within a population. Thus, for some chromosomal fragments, the direction of fork migration during
DNA synthesis may change from one cell division to the
next (Fig. 2).
Individual replication origins fire at characteristic and
reproducible times during S-phase. For example, the yeast
origins ARS305 and ARS607 fire shortly after cells have
entered S-phase and are considered ‘‘early’’ origins, whereas
the yeast origin ARS1 fires during the first half of S-phase
but several minutes after ARS305 [1]. Replication origins
such as ARS501 or the cluster of origins on chromosome
XIV (ARS1411, 1412, 1413 and 1414) fire near the end of Sphase and are considered ‘‘late’’ origins [7,8]. A recently
constructed temporal replication map for the yeast genome
Fig. 1. The diagram depicts the temporal replication pattern of yeast chromosome VI showing many but not all of the origins on this chromosome. Origins near
the centromere fire earlier than those near the ends of this chromosome. For a more complete description of the replication of this and other yeast chromosomes
that focuses on distilling information from the genome wide studies [1,2], see the short review by Newlon and Theis [70].
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Fig. 2. Competition between two closely spaced origins can influence origin efficiency and the physical replication pattern of the chromosomal region between
the two origins (*). (A) In this diagram, origin A usually fires earlier than origin B. Thus origin B is inefficient because a fork established at origin A replicates
the DNA region containing origin B before origin B fires. (B) If origin A is rendered non-functional by creating a mutation in a DNA sequence element
necessary for origin function, origin B now fires efficiently. Note that the molecular history for each daughter chromosome has changed.
indicates that in general, most early origins are positioned
toward the central portion of yeast chromosomes, while late
firing origins are positioned nearer the telomeres such that
the central portion of chromosomes replicate before the ends
[1] (Fig. 1).
There is not a consistent relationship between replication origin efficiency and the time at which an origin fires
during S-phase—some late firing origins are efficient and
others are not [9,10]. Some late origins are inefficient
because they are located near another earlier firing origin.
The earlier origin establishes a replication fork that replicates the later origin before it has a chance to fire [11].
Presumably, this inactivates the competent initiation complex assembled at the late origin and the mechanisms that
prevent re-replication of DNA during a single eukaryotic
cell cycle prevent the late origin from acquiring the
capacity to fire until the next S-phase (Fig. 2A and Ref.
[12]). Direct experiments support a role for such an
‘‘origin interference’’ mechanism in origin efficiency
[13 –15]. Specifically, if an earlier firing neighbor origin
is inactivated, the normally inefficient origin initiates more
frequently during S-phase, but the time at which it fires
does not change (Fig. 2B). Other origins, regardless of the
time during S-phase when they fire, may be inherently
inefficient because they fail to assemble the multi-protein
complex necessary to mark them as replication origins [2].
Several independent mechanisms may affect origin efficiency or timing including DNA sequence features intrinsic to the particular origin. For many origins it is clear that
chromosome context and/or specialized chromatin structures play a role (Section 2).
1.3. Mechanism of replication initiation
1.3.1. Sequence requirements of replication origins in S.
cerevisiae
The first yeast origins were identified as small chromosomal regions (DNA elements of a few hundred base pairs)
that gave extra-chromosomal circles (plasmids) the ability to
replicate autonomously [16]. Later experiments using molecular techniques for direct origin mapping [17] indicated
that these autonomous replicating sequences (ARS) coincided with bona fide replication origins in their native
chromosomal contexts. Because of this history, origins in
yeast are named ARS followed by a number that most often
designates their chromosomal position.
Only a few individual ARS elements among the ~400
have been characterized systematically by comprehensive
linker-scan mutational analysis, so it is not possible to
describe a single common organizational structure for a
yeast origin. However, the analyses that have been completed suggest that many origins may share some common
features, including a conserved A-element and B1-element
that form the Origin Recognition Complex (ORC) binding
site (Fig. 3A). ARS1 has been particularly well characterized
in terms of activity on plasmids, on the chromosome and
ORC binding in vitro [15,18,19]. ARS1 is f 150 bp and
consists of four functionally defined modular elements: an
A-element found in most yeast origins, including ARS305
and ARS307[20 –22], and a series of B-elements, B1, B2
and B3 that lie 5V of the A-rich strand of the A-element
[18]. Mutation of the A-element in any of the ARSs shown
in Fig. 3A abolishes origin activity. However, the A-element
M. Weinreich et al. / Biochimica et Biophysica Acta 1677 (2004) 142–157
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Fig. 3. (A) The organization of three ARS elements defined by comprehensive linker-scan analyses [18,20,22]. The ARS core consensus sequence (ACS)
required for ORC binding has been determined by examining a large number of yeast origins (see text Section 1.3.1 and Ref. [139]) and is also shown. (B) A
depiction of the ‘‘four phases’’ of origin activity as described in the text (Section 1.3.2).
is not sufficient; each of these origins also requires at least
one other B-element for origin activity. However, in contrast
to the A-element, the B-elements vary considerably between
origins in terms of their sequences, number and distance
from the A-element (Fig. 3A). Studies of ARS1 indicate that
ORC contacts both the A- and B1-elements and it is
probable that and A- and B1-element together form a
bipartite recognition site for ORC in most origins [23 –
25]. The ARS1 B2-element may contribute to the loading of
the six-subunit MCM (mini-chromosome maintenance) helicase. In ARS1, the B3-element contains a binding site for the
transcription factor Abf1p; many origins do not contain
Abf1p binding sites, including ARS305 and ARS307, but
may contain binding sites for other as yet unidentified
factors [20 – 22].Finally, although the A-element is conserved in most replication origins, some origins contain
multiple near matches to the A-element that must be
collectively mutated to abolish origin activity [26]. These
‘‘compound’’ origins have not been characterized as systematically as the ARS elements pictured in Fig. 3A and so
their organization is less clear. However, their existence
indicates that not all origins in yeast necessarily share the
structure suggested by the ARSs in Fig. 3A.
The sequences required for replication initiation are
termed replicators whereas the positions at which replication
initiates (origin unwinding and priming of DNA synthesis)
are termed origins. A PCR-based technique called Replication Initiation Point (RIP) mapping can map the position of
the start of leading strand synthesis to a single nucleotide
[27]. RIP mapping experiments of ARS1 suggest that in
yeast, replicators and origins are essentially coincident. For
ARS1, leading strand synthesis begins at a single nucleotide
between the B1- and B2-elements [28]. Recent experiments
with a limited number of origins in other organisms indicate
that leading strand synthesis occurs at a discrete position
near the binding site for that organism’s ORC [29]. Thus,
ORC may mark the site of origin unwinding and the start of
leading strand synthesis in all eukaryotic organisms. However, it is important to note that only a limited number of
origins have been examined and the sequences required for
full replicator function may lie some distance from the
origin site for some origins [30 –33].
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1.3.2. Proteins and steps required for replication initiation
A current model for replication initiation at a yeast origin
is shown (Fig. 3B). Experiments performed in a variety of
organisms indicate that the proteins and steps required for
initiation are largely conserved in eukaryotes even though
the sequence elements required for origin activity are not.
(For comprehensive reviews see Refs. [4,34].)
The replication initiation cycle can be divided into at
least four phases. The first is formation of an origin
DNA – ORC complex [19]. ORC binds to an origin via
direct protein –DNA contacts between at least four of its
six different subunits and nucleotides present in both the
A- and B1-elements [24]. ORC binds ATP and is an
ATPase, and origin binding by ORC requires ATP binding
but not hydrolysis [19,35]. In vivo footprinting and chromatin binding experiments indicate that ORC is bound to
its target sites throughout most if not all of the cell cycle
[36,37]. Thus, one of ORC’s major roles is to mark origins
as the sites of replication initiation. ORC also recruits
additional initiation proteins during G1 that together form
the pre-replicative complex (pre-RC), a multiprotein DNA
complex necessary for origin firing. ORC may have roles
in the subsequent events of replication initiation [38,39]
including later steps that contribute to elongation and
genome integrity [40].
In the second phase, ORC promotes assembly of a
multiprotein complex called the pre-replicative complex
(pre-RC) at origins. The pre-RC was defined operationally
as the change in the in vivo footprint that can be detected at
origins during the G1 phase [36]. Formation of the pre-RC
requires minimally ORC, Cdc6p, Cdt1 and the MCM
complex [4]. Cdc6p directly interacts with ORC and is
required together with Cdt1p to ‘‘load’’ MCM, the putative
replicative helicase, at the origin in a reaction that requires
ATP [41, 42].
In the third phase, additional proteins join the pre-RC
immediately prior to replication initiation. These include
Cdc45p, the recently described four-subunit GINS complex
and the replicative DNA polymerases [4,43]. Cyclin-dependent kinase (CDK) activity is required for Cdc45p to
associate with the pre-RC [44], and the association of
Cdc45p with origins coincides with their time of activation—late origins recruit Cdc45p later in S-phase than
earlier origins [45]. The Cdc7-Dbf4p kinase also associates
with chromatin just prior to S-phase and interacts with ORC
[46,47]. Cdc7-Dbf4p may activate the MCM helicase or the
Pola-primase at origins. These sequential steps involve at
least 20 different polypeptides that must assemble coordinately within a chromatin context at hundreds of replication
origins throughout the genome for S-phase to commence.
The fourth phase, origin firing, signals the beginning of
S-phase, and involves localized DNA unwinding at the
origin and the start of DNA polymerization. The mechanism
of origin unwinding and the initiation of DNA synthesis are
not understood at eukaryotic origins and may occur simultaneously with the association of some of the factors out-
lined in phase three. For instance, phosphorylation catalyzed
by the Cdc7-Dbf4 kinase may promote conformational and/
or polypeptide changes within the multiprotein complex
bound to origins that are necessary for DNA unwinding
and the start of DNA polymerization. Finally, CDK activity,
which remains high during the S- and G2/M-phases, negatively regulates ORC, MCM and Cdc6p to prevent the
assembly of new pre-RCs [12,48,49,50]. Therefore, CDK
activity is required both to activate the pre-RC’s by promoting the binding of Cdc45p (third phase) and simultaneously prevent new pre-RC assembly (and re-replication
within a single S-phase) until the next G1 phase.
In summary, replication initiation at a given origin is a
complex process requiring multiple proteins binding in
several steps. Each individual step could conceivably be
enhanced or inhibited by particular chromatin environments.
1.4. Why so many replication origins?
The number of replication origins per chromosome is not
merely a consequence of the larger genomes in eukaryotes.
S. cerevisiae has a 12-Mb genome distributed over 16
chromosomes, and therefore each single yeast chromosome
is considerably smaller than the 4.6-Mb E. coli genome. Yet
yeast replication origins occur on average every 20 to 40 kb,
a hundred times more densely distributed that one would
predict by comparison to the E. coli genome. The vast
difference in fork migration rates between eukaryotes and
prokaryotes may explain in part the need for multiple
replication origins per eukaryotic chromosome. DNA replication forks migrate at rates about 30 times slower (fork
migration rates of f 3 kb/min [1,50] in yeast compared to
E. coli (fork migration rates of f 100 kb/min [51]), yet
under optimal growth conditions DNA synthesis periods are
comparable ( f 40 min) and yeast cells divide only four to
five times more slowly than E. coli. Eukaryotic chromatin is
often invoked as an explanation for the slow migration rate
of DNA replication forks, but a direct experimental test of
this idea is not yet possible. In particular, chromatin templates that recapitulate the complexity of biological chromatin are still in early stages of development and structural
characterization [52,53]. In addition, the bacterial chromosomes are also packaged with and condensed by various
proteins. Although these proteins differ from the highly
conserved and well-described eukaryotic histone octamer, it
is difficult to imagine that the condensation of the prokaryotic genome might not present a similar challenge to
migration of a replication fork. Thus, it is not clearly
established whether or how chromatin structure itself may
slow replication fork migration in eukaryotes. Regardless,
the use of multiple initiation events per chromosome probably compensates for slower fork migration rates in maintaining an efficient rate of genome duplication and S-phase
progression in eukaryotic cells.
Despite the contribution that multiple origins per chromosome may make to efficient genome duplication in S.
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cerevisiae, there are still many more replication origins than
necessary. For example, based on the values discussed
above, S. cerevisiae would need about 100 replication
origins to duplicate its genome at a rate sufficient to
accommodate its S-phase, about four times less than the
current estimates for origin numbers in this organism [1,2].
In addition, direct experimental evidence indicates that yeast
chromosomes have more origins than they need for their
timely replication during the S-phase; several origins on
chromosome III can be deleted without substantially affecting the ability to inherit faithfully this chromosome during
cell division [54]. Therefore, for the purpose of genome
duplication, yeast replication origins are redundant. Such
redundancy might permit individual origins to evolve more
specialized functions within chromosomes that are distinct
from a dedicated role in DNA synthesis.
Although there is no compelling evidence that eukaryotic
replication origins have any direct role beyond initiating
DNA synthesis, it is clear from studies in several organisms
that one of the central and conserved protein complexes
required for origin activity, ORC, has evolved to perform
functions distinct from its role in DNA synthesis [38]. For
example, in budding yeast, ORC has an important role in
establishing chromosomal domains of repressive (silent)
chromatin at the cryptic mating-type loci [55]. ORC interacts with Heterochromatin Protein 1 (HP1) in both Drosophila and Xenopus and is concentrated within defined
heterochromatin regions in the Drosophila genome [56].
The human Orc6p subunit is localized to kinetechores and
the cleavage furrow during M-phase and so may have roles
distinct from its role in replication in human cells [57]. Thus
ORC, and perhaps other origin proteins, have non-DNA
synthesis roles in regulating chromatin structure in yeast and
metazoans.
Replication origins may also have important albeit indirect non-DNA synthesis roles in the genome by virtue of
their central role in establishing the temporal and physical
genome replication maps. In particular, studies of S. cerevisiae indicate that the temporal and physical pattern of
chromosome replication is established primarily by the
activity of replication origins [1], indicating the potential
to alter a chromosome’s replication map by controlling
origin activity. In terms of the temporal map, the time
during S-phase when a chromosomal region is replicated
correlates strikingly with chromatin structure and gene
activity in many organisms [58]. Transcriptionally inactive
genes and/or heterochromatin are often the last portions of
the genome to be duplicated. This is perhaps because origin
activation within heterochromatin is delayed and therefore a
fork that came from a distant origin replicates heterochromatin. Even in budding yeast the rare portions of the
genome controlled by chromatin structures analogous to
multicellular heterochromatin are extremely late replicating
[1]. A recent chromosome-scale study of replication timing
in Drosophila melanogaster Kc cells reveals a significant
correlation between late replication and transcriptional qui-
147
escence [59]. However, this correlation is not absolute either
in Drosophila or other organisms [59 – 61]. Further examinations of the relationship between origins, replication
timing and chromatin structure will clarify the view of the
mechanisms of and purpose for a defined temporal map of
genome replication in both microbes and metazoans.
Initiation at an individual origin establishes a defined
asymmetric physical pattern of replication to flanking chromosomal regions because of the inherent 5V to 3V polarity of
DNA synthesis. A fragment replicated by a fork moving
from a given origin gives rise to one daughter molecule as a
result of lagging-strand synthesis and another daughter
molecule as a result of leading-strand synthesis. Thus, the
two daughter molecules, although identical in sequence,
have a different molecular history (Fig. 2). This naturally
arising asymmetry between daughter molecules is implicated in regulating mating-type switching in S. pombe [62,63]
and contributes to strand bias in mutagenesis in S. cerevisiae[64]. Regulating the activity of individual origins is an
obvious mechanism to change the direction from which a
particular chromosomal region is duplicated, and the different physical replication patterns that would result could
affect gene expression and differentiation through chromatin
structure if particular chromatin assembly or modification
pathways were differentially partial to DNA arising from
either leading- or lagging-strand synthesis [63,65,66].
Another simple consequence of having multiple origins
is the ability to control the rate of the cell division cycle by
controlling the number of active replication origins. This
could be important in multicellular organisms. In particular,
during embryogenesis of multicellular organisms, the earliest cell cycles are rapid and may depend in part on the
ability to use many sequences as replication origins [67 –
69]. By reducing the number of active origins per chromosome later during development, the S-phase and hence the
entire cell division cycle could be reduced to a rate more
compatible with cell differentiation that requires the synthesis of more specialized and complex cellular structures. The
possible relationship between reducing the number of origins and developmental fates remains to be established.
2. Chromosomal context and chromatin structure
modulate origin activity
This portion of the review is divided into four subsections, each of which focuses on different evidence
supporting roles for chromosome context or chromatin
structure in the activity of budding yeast replication
origins. In Section 2.1 we will review the evidence for
chromosomal position effects on the activation time and
efficiency of yeast replication origins. In Section 2.2 we
will summarize recent studies that implicate specialized
chromatin proteins in controlling a subset of origin position effects. In Section 2.3 we will discuss a recent study
that provides evidence that the acetylation state of nucle-
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osomes surrounding an origin can regulate origin activation time. In Section 2.4 we will present selected studies
that provide evidence for nucleosome positioning in controlling cellular origin activity.
2.1. Chromosome position influences the efficiency and
timing of origin activation
A number of observations indicate that origin activity in
yeast is subject to a position effect that resembles classic
chromatin-mediated position effects on gene transcription.
An early indication of this phenomenon was that some
sequences that provide ARS activity to plasmids (and thus
function well as replication origins in this context) fail to
function as chromosomal origins in their native context
[11,70,71]. Thus, a DNA sequence element with the inherent capacity to function as an origin can be completely
inhibited in particular chromosomal environments. An example of a chromosomal region capable of exerting such a
position effect is the cryptic mating-type locus HML—a
normally active origin (ARS305) placed within HML
becomes inactive [72].
Position effect phenomena can also account for the
activation time of efficient but late firing origins. For
example, ARS501 is an efficient late origin in its chromosomal context but when it and the surrounding f 15 kb of
chromosomal sequence are moved to a plasmid, the ARS501
origin fires early [7]. Thus, sequences many kilobases distal
from ARS501 are somehow influencing the time during Sphase at which this origin fires. Significantly, moving an
earlier firing origin, ARS1, near ARS501 on the chromosome
causes ARS1 to fire later and at a time similar to origin
activation for ARS501. The position of ARS501 relative to
the telomere (27 kb from the right telomere of chromosome
V) is important, indicating that telomeres can exert fairly
long-range position effects on origins [73]. As stated earlier,
based on whole-genome replication timing analysis in yeast,
origins located proximal to telomere ends ( f 40 kb or
closer) are often activated later in S-phase [1]. Recent
studies have identified proteins required for the telomereposition effect (TPE) on replication origins (Section 2.2).
ARS501 is a late replication origin because of its proximity to telomeres, but the cluster of late origins on
chromosome XIV (ARS1411, 1412, 1413 and 1414) are
over f 150 kb from the telomere. Also in contrast to
ARS501, the late activation time for at least two of these
origins, ARS1412 and ARS1413, can be recapitulated in a
plasmid context [8], supporting the idea that their late
activation is telomere-independent. However, in the case
of both ARS1412 and ARS1413, considerable amounts DNA
sequence flanking the intrinsic origin sequences are required
to fully recapitulate the late origin activation time, and it
appears that there is not an easily identifiable discrete
sequence element that determines their late activation. Thus,
for both a telomere-proximal late origin and internal late
origins, large chromosomal regions distinct from the intrin-
sic ARS elements are required for determining their late Sphase activation.
Although most of the existing data for inefficient and/or
late activating origins support a role for large regions
external to the origin in controlling the origin’s distinguishing activation properties, one recent exception is worth
noting. In particular, studies of one extremely late activating
origin, ARS301, suggest that this origin is inefficient on the
chromosome regardless of its position [72]. ARS301 is one
of the few origins in yeast closely associated with elements
called silencers present at the yeast cryptic mating-type loci
HMR and HML and necessary for assembling the heterochromatic-like state at these loci (reviewed in Ref. [74]). In
its normal location at HML, ARS301 is inactive in part
because it is programmed to fire so late that it is replicated
by a replication fork from a more distant origin before it
ever has a chance to fire [75]. However, ARS301 functions
as an origin in a plasmid context, although it is important to
note that even in this context it is inefficient. HML can exert
a position effect on an origin—substituting ARS301 with the
active and early firing ARS305 at HML causes ARS305 to
become chromosomally inactive. And yet, surprisingly this
position effect cannot explain fully the late and inefficient
activity of ARS301. Specifically, replacing the active and
early firing ARS305 with ARS301 does not lead to early and/
or efficient activation of ARS301. Rather, ARS301 remains
inactive or, in the appropriate mutant backgrounds that
allow extremely late origins to fire [76], extremely late
firing. Thus, for some origins intrinsic sequences may also
contribute to their efficiencies or activation times [72].
Even if intrinsic sequences contribute some of the
distinctive features to individual origins, the significant
effect of chromosomal context on origin activation is well
established. The phenomenon is so reminiscent of classic
chromatin-mediated position effect on transcription that it is
not surprising that some of the proteins required for position
effects on transcription in yeast also have effects on origin
activation (Section 2.2).
2.2. ‘‘Specialized’’ chromatin structures and origin
activation
2.2.1. The Ku proteins are required for late activation of
ARS501 and other telomere-proximal origins
A recent study provides evidence that the telomerebinding Ku protein complex (Ku) controls the late activation
of the telomere-proximal ARS501 origin and probably other
telomere-proximal origins [77]. Ku has critical functions in
the repair of double-stranded DNA breaks and also binds and
functions at telomeres, the defined ends of linear eukaryotic
chromosomes [78,79]. Significantly Ku, consisting of the
yeast Ku proteins yKu70 and yKu80, also contributes to the
specialized chromatin structure that forms at telomeres and
causes a TPE on gene transcription [80 – 82]. TPE describes
the phenomenon whereby RNA polymerase II genes placed
within several kilobases of telomeres are repressed by a
M. Weinreich et al. / Biochimica et Biophysica Acta 1677 (2004) 142–157
mechanism akin to position-effect variegation in Drosophila
[83]. TPE requires a number of different proteins including
three of the silent information regulator (SIR) proteins
(Sir2p, Sir3p and Sir4p) initially identified for their roles in
repressing copies of mating-type genes present at the silent
mating-type loci HMR and HML [84]. Ku is required for TPE
because it helps recruit the Sir proteins that function together
to assemble a heterochromatin-like structure near the ends of
chromosomes [82,85]—the SIR-dependent heterochromaticstate nucleates at telomeres and ‘‘spreads’’ into the chromosome up to 10 kb [86,87]. ARS501, about 27 kb from the
telomere is beyond the limit of the SIR-dependent TPE and
yet its late activation is telomere position-dependent.
In a yeast strain containing a deletion of the yKu70 gene
the origin activation time for ARS501 shifts to a significantly earlier time in S-phase [77]. This deletion also
advances the replication times for other regions near
telomeres but not the internally positioned chromosome
XIV ARS cluster, consistent with a role for Ku in controlling the late activation time of telomere-proximal origins.
One postulate is that Ku70/Ku80 contributes to a specialized chromatin environment near chromosome ends that
extends into the chromosome 30– 40 kb from the telomere
that may require association with the nuclear periphery,
another property of telomeres that requires the Ku complex
[80]. And yet this chromatin structure is distinct from the
SIR-dependent chromatin structure at the ends of telomeres
that also requires the Ku complex. Specifically, a deletion
of SIR3 has little effect on the origin activation time of
ARS501 even though it abolishes TPE and does contribute
to the activation of origins within the zone that is subject to
SIR-dependent TPE [88]. Thus, the Ku-mediated position
effect on late origin activation is exerted over a large
distance from telomeres ( f 35 kb) and in this regard is
distinct from the SIR-dependent chromatin-mediated TPE
that is exerted over a shorter distance ( f 10 kb) from
telomeres.
2.2.2. SIR effects on origin activation
As stated above, the Sir proteins function in the assembly
of specialized chromatin structures analogous to heterochromatin. Several recent studies indicate that SIR-dependent
chromatin can affect the activity of replication origins.
Before discussing these studies, we present a brief summary
of SIR-dependent chromatin formation. Several recent
reviews offer more detailed and comprehensive information
on this subject [89 – 91].
SIR-dependent chromatin structures, also referred to as
silent chromatin, repress the transcription of fully intact
RNA polymerase II transcribed genes. Silent chromatin
forms at three different regions in the yeast genome
(reviewed in Ref. [91]): (1) the ends of telomeres, (2) the
nucleolar rDNA locus and (3) the cryptic (silent) matingtype loci HMR and HML. All three types of silent chromatin
require the Sir2p, the founding member of a conserved
family of NAD-dependent protein deacetylases [92]. Cur-
149
rent models propose that Sir2p deactylates nucleosomes
within all three loci and that this modification contributes
directly to the formation of a heterochromatin-like structure.
All three forms of SIR-dependent silent chromatin require a
nucleation event whereby specific multi-protein – DNA
complexes recruit the SIR protein(s) to the locus. The
recruitment proteins and precise interactions necessary for
nucleation differ at the three different regions [93]. After the
nucleation step, the silent state physically ‘‘spreads’’ from
the nucleation site to assemble a larger domain of silent
chromatin encompassing an array of nucleosomes. At telomeres and the HM loci, the spreading mechanism appears
similar—current models posit that Sir2p deacetylates Nterminal histone tails within a nucleosome near the nucleation site, Sir3p then binds the deactylated nucleosome [94]
and recruits an additional complex of Sir2p and Sir4p and so
on until an entire array of nucleosomes is encompassed into
the silent state [91,95]. At rDNA, Sir3p and Sir4p are not
required for the silent chromatin-state but Sir2p certainly is.
Remarkably, the directional movement of the RNA polymerase I machine that transcribes the rDNA in this locus is
also required for the SIR2-dependent spreading of the silent
state [96]. Regardless of the precise mechanisms, all three
silent regions require the deacetylase activity of Sir2p and
additional proteins that assemble a larger domain of higherorder repressive chromatin.
Strong evidence for a SIR-chromatin effect on origin
activity of ARS elements associated with telomere-repeat
sequences comes from a study focused on understanding
the late replication time of telomeres using the right
telomere of chromosome V (ARS501, discussed above, is
f 35 kb from this telomere) [88]. Telomeres consist of
highly repetitive sequences (telomere repeat sequences) at
their extreme ends—these are the sites of SIR-nucleation.
Next to these lie sub-telomeric repeats, the X and YV
elements. Each of these elements contains an ARS but this
study demonstrates that the X element ARS on chromosome V is an inactive origin due to the SIR-dependent
chromatin state in this region. Because of the repetitive
nature of the X and YV regions and their presence at most
telomeres, it is not simple to examine X or YV origin
activity directly by 2-D origin mapping. However, the
region just next to these repeats is unique and, strikingly,
in a strain containing a deletion of SIR3, this region
replicates 9 min earlier than in a SIR3+ strain. Several
careful experiments provide compelling evidence that this is
because the YV ARS is activated earlier during S-phase and
the X ARS is now an active origin. In contrast, the
activation time of ARS501 appears unaffected by a deletion
of SIR3. If ARS1 is engineered next to telomeric repeats
(essentially replacing the YV and X elements) it fires less
efficiently and much later than it does at its native position,
but again, deletion of SIR3 allows earlier and more efficient
activation of ARS1 at this location. Thus the SIR3 effect on
origin activity is not origin-specific as expected for a factor
that mediates a position effect.
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Together the SIR3 and Ku study let us picture two
mechanisms that control telomere-associated position effect
on origin activity. First, origin activation for origins close to
the telomere (within 10 kb) is probably controlled by the
same SIR-dependent position effect responsible for TPE.
Second, the telomere-end binding protein complex Ku
additionally controls the late activation of more distal
telomere-associated origins (35 – 40 kb) by a SIR-independent mechanism that may require chromosome association
with the nuclear periphery.
Another recent independent study indicates that the
HMR-E silencer, which is the silencer that nucleates the
assembly of SIR-dependent silent chromatin at HMR can,
when placed in two tandem copies near the active early
origin ARS305, cause ARS305 to fire late [97]. The late
activation of ARS305 requires the SIR1 gene, the only SIR
whose function in silent chromatin is confined to the HMR
and HML loci. In addition, the late activation time of
ARS305 correlates with the ability of either the two copies
of HMR-E or a tethered Gal4-Sir4p to transcriptionally
silence an adjacent gene. Thus SIR-dependent chromatin
can exert a position-effect on both transcription and origin
activation.
The final SIR effect on origin activity we will discuss is
the effect of SIR2 on origin activation within the rDNA
cluster [98,99]. As discussed above, a type of silent chromatin forms within the rDNA cluster that can repress
heterologous RNA Polymerase II genes placed between
rDNA genes, but this chromatin is distinct from the SIRdependent chromatin at the HM loci and telomeres in that it
requires only one of the SIR genes, SIR2. Nevertheless, SIR2
is required for regulating both the position effect on transcription and origin activation within the rDNA and so these
data are most appropriately discussed here.
The rDNA locus is located within the nucleolus and
contains 100– 200 copies of a 9.1-kb repeat that encodes the
5S and 35S rRNA genes that are divergently transcribed by
RNA polymerase III and RNA polymerase I, respectively.
Each repeat contains an ARS element located between the
two rRNA genes and a replication fork barrier is located to
the left of the ARS element such that forks can only proceed
to an adjacent repeat in the rightward direction. Only 20% of
the ARS elements are active in any given cell cycle,
indicating that some mechanism is preventing the activation
of many origins (reviewed in Ref. [99]).
A recent study that uses the technique of molecular
combing to examine replication of individual DNA strands
indicates that SIR2 inhibits the activation of many origins
within the rDNA repeats [98]. In wild-type (SIR2) cells,
only 25% of the origins are active within a single cell cycle
as measured by molecular combing. In addition, this single
molecule technique indicates no epigenetic control of the
origins that fire within successive cell cycles—that is, an
origin active in one cell cycle might be inactive in the next
and vice versa. Large gaps (>60 kb) are present between
actively replicating DNA in early S-phase, indicating that
active origins are separated by several inactive origins. In
contrast, in the absence of SIR2 (sir2D) almost twice as
many origins are activated within the array. Thus, although
SIR-mediated position effect within rDNA may employ
different mechanisms than SIR-mediated silencing at the
HM loci and at telomeres, it shares the ability to inhibit the
activity of local origins.
Based on the association between SIR-mediated chromatin and the inefficiency or delay in origin activation, it
is somewhat surprising that the inactive origins present at
the HM loci are not subject to SIR-control. Both HMR
and HML contain ARS elements that are closely associated with the silencer elements that nucleate the assembly of
SIR-dependent silent chromatin at these loci. As described
above, at HML the ARS elements are inactive on the
chromosome but are functional as plasmid origins. The
HMR silencers are associated with ARS elements that act
as extremely inefficient chromosomal replication origins
[100,101], and the HMR-E silencer-associated origin fires
late in S-phase in the few cells in which it is active [102].
Therefore, both the HML and HMR loci are replicated late
during the S-phase, primarily by forks coming from
distant origins. Interestingly, however, and in contrast to
the effect of SIR-dependent chromatin on the sub-telomeric repeat ARSs, the origin activity at HMR and HML
is not affected dramatically by mutations in SIR genes. In
a strain lacking SIR4, HML origins remain inactive even
though the SIR-dependent chromatin state, as measured by
gene activation and chromatin-binding experiments on the
other Sir proteins [71], is abolished. Similarly, in a strain
lacking SIR2, the HMR-E origin activity is only slightly
enhanced and the locus is still replicated late in S-phase
[101,102]. Thus, other features at these loci contribute to
the inefficiency and late activation of their associated
replication origins, possibly even features inherent to the
sequences in these regions. Given that these loci are
located near the ends of chromosome III but out of the
range of the TPE, it is possible the yeast Ku proteins
contribute to the late replication of these loci. Regardless,
it is noteworthy that SIR-mediated chromatin can affect
replication origin activity in some but not all contexts in
which it forms.
The mechanisms by which SIR-mediated chromatin can
inhibit origin activity are unknown. However, the Sir2p is a
protein deacetylase, and recent evidence indicates that
histone acetylation can promote origin activity (Section
2.3). Thus, the SIR-mediated effect on origin activity may
reflect a more general role for the acetylation state of
histones surrounding origins on the efficiency and timing
of their activation. Intriguingly, recent studies indicate that
the Sir2p deacetylase may inhibit origin activity on a more
extensive scale that includes origins outside of the rDNA
and telomeric domains, raising the possibility that Sir2p
itself might have more global roles in negatively regulating
replication initiation (Donald Pappas and M. Weinreich,
unpublished observations).
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2.3. Histone acetylation advances the activation time of
origins
Studies in the past decade have established a role for
posttranslational histone modifications in the control of
eukaryotic transcription in eukaryotes (discussed in Refs.
[103,104]), and a recent study indicates that such modifications will also have critical roles in origin activation.
Specifically the acetylation state of nucleosomes surrounding origins affects their activation time during S-phase, with
increased levels of acetylation advancing origin activation
time [105].
The first clue that the acetylation state of nucleosomes
might influence origin activation came from studies of the
role that one of the major histone deacetylases, Rpd3p,
plays in the histone acetylation state of the yeast genome.
Genome-wide studies show that deletion of RPD3 results
in global changes in both histone acetylation and the
steady-state mRNA levels of over 400 genes, consistent
with the now well-established importance of these modifications in transcription [103,104]. Notably, in the absence of RPD3, the acetylation state of many intergenic
chromatin regions increases [106]. Since most origins are
also intergenic [2,107], it follows that the acetylation state
of chromatin surrounding many origins should increase in
cells lacking RPD3 and indeed chromatin immunoprecipitation experiments indicate that this is true for several
individual origins [105]. Even more intriguingly, the origin
activation time for each of these origins advances to an
earlier time after the cells lacking RPD3 are released from
a G1-cell cycle arrest, and the extent of advancement
correlates well with the extent of increased acetylation.
For example, in a strain lacking RPD3, the acetylation
state of chromatin surrounding both ARS603 and ARS305
increases, but the increase is greater for ARS603. In turn,
the activation time for both origins advances relative to the
wild-type strain but the activation time for ARS603 advances to a greater degree. Consistent with the idea that
Rpd3p deacetylates chromatin surrounding a large number
of yeast origins, replication of the entire genome is
advanced in a strain lacking RPD3. For example, 20 min
after release from G1 arrest, few of the cells from a
RPD3+ strain have acquired a 2C DNA content but one
third to one half of the cells from a strain lacking RPD3
have. Normally, late replication origins, such as those in
the ARS chromosome XIV cluster, may be particularly
sensitive to genome acetylation status controlled by RPD3;
such late origins behave as bona fide early origins in
strains lacking RPD3 because they now escape the checkpoint inhibition of late origins in the presence of replication stress normally exerted by Rad53p [76] (Oscar
Aparicio, personal communication).
These observations provide compelling evidence for a
role for Rpd3p and the acetylation state of chromatin in
controlling origin activation time on a rather global scale,
but they do not address whether this control is direct. For
151
example, the mRNA levels for hundreds of genes change
in strains lacking RPD3. One might expect a similar effect
on global origin activation even if only one of these genes
encodes a protein that is limiting for origin activation. In
addition, TAP-tag purification experiments suggest that
Rpd3p and Cdc45p are in a common protein complex
(http://www.yeast.cellzome.com). Since Cdc45p must bind
origins prior to firing, one possibility is that Rpd3p
directly inhibits Cdc45p and delays its association with
origins and consequently origin activation. However, an
experiment that targets increased acetylation to a single
specific origin argues against these interpretations and for
a more direct role of histone acetylation in enhancing
origin activation time [105]. Specifically, targeting the
Gcn5p histone acetyl transferase (HAT) to a region near
the late origin ARS1412 increases the level of acetylation
of the chromatin surrounding ARS1412. This engineered
version of ARS1412 with locally enhanced chromatin
acetylation fires up to 20 min earlier than the normal
ARS1412 from an otherwise identical yeast strain and,
importantly, this earlier firing depends on the presence of
GCN5, indicating that it is not a technical consequence of
the DNA sequence changes near ARS1412 required to
recruit this HAT. Together with the analysis of replication
in strains lacking RPD3, this study provides compelling
support for the view that the acetylation state of nucleosomes around an origin’s local environment has a direct
effect on that origin’s activation time and that the Rpd3p
deacetylase is a major negative regulator of origin activation in yeast.
The next level of experiments related to the role of
nucleosome modifications in origin activation should
delve into the types of chromatin modifications most
permissive (or non-permissive) to origin activation and
the steps of origin activation that are sensitive to an
origin’s chromatin neighborhood. For example, are there
specific patterns of histone acetyation that are particularly
crucial to origin activation and are such patterns cell
cycle-regulated? In terms of steps in origin activation that
may be affected by chromatin, one hint may come from
analysis of the Cdc45p’s association with origins [45]. As
stated in an earlier section, origins recruit the Cdc45p at a
time that correlates with their activation. And in strains
lacking RPD3, Cdc45p loading is temporally advanced
along with origin activation [105] (Oscar Aparicio, personal communication). Thus, Cdc45p loading that is
essential for initiation may be a step that is directly
enhanced by acetylated nucleosomes, but the mechanism
by which this enhancement occurs is unknown. It is
possible that acetylated nucleosomes around origins directly enhance the accessibility of an initiation protein,
such as MCM, to Cdc45p or that they bind Cdc45p
directly. However, it is possible that nucleosome acetylation enhances the binding or modification of another
factor that in turn helps recruit Cdc45p. Reconstituting
pre-RC assembly in the context of defined nucleosome
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arrays may help address this issue, as will continual
refinement of our understanding of the factors and modifications leading to changes in the pre-RC that occur just
prior to initiation.
2.4. Nucleosome positioning and origin activity
In addition to histone modification, nucleosome movement and positioning, and the ATP-dependent complexes
that can move nucleosomes and alter chromatin structure,
have emerged as major factors in transcription regulation
[108 –110]. Several studies indicate that nucleosome positioning may regulate origin activity as well [111 –113].
Studies of ARS1 indicate that both the Abf1p binding site
and ORC binding site (A-element) of this origin act as
nucleosome boundaries, positioning nucleosomes outside
of and flanking this origin. Mutations in the Abf1p
binding site increase the chance that a nucleosome will
form over the A-element and reduce ARS1 origin activity
[114]. Similarly, engineering a specific protein-binding site
outside of ARS1 that results in positioning a nucleosome
over the otherwise functional A-element reduces origin
activity of ARS1 [112]. These data indicate that ARS1 is in
a nucleosome-free zone and further that nucleosomes
formed over essential origin sequences can inhibit origin
activity.
A more recent biochemical study of ORC and Abf1p
binding to ARS1 assembled into chromatin in an in vitro
chromatin assembly extract confirms and extends these
findings [113]. In particular, ORC and Abf1p bind to their
target sites in ARS1 and precisely position nucleosomes
around ARS1 in a pattern that recapitulates the nucleosome
positioning observed at the ARS1 origin in vivo. Thus, the
nucleosome-free zone of yeast ARS1 is recapitulated in a
heterologous chromatin assembly extract (Drosophila S190
extract [115]) in the presence of purified ORC and Abf1p—
no other yeast factors are required. In vivo, an intact Aelement is required in both ARS1 and another origin,
ARS307, to prevent a nucleosome from encroaching on
the origin sequences. These in vitro and in vivo data confirm
the inhibitory role for nucleosomes in origin activity and
indicate roles for both ORC and Abf1p as potent nucleosome boundary or positioning factors.
Additional experiments provide evidence for a positive
role for nucleosomes positioned outside but near the Aelement of ARS1 by the binding of ORC [113]. Specifically, if a sequence-specific DNA protein-binding site is
introduced 3V of the A-rich strand of the A-element of
ARS1, the nucleosome normally positioned by ORC adjacent to the A-element is displaced away from the origin in
vivo. This displacement correlates with a reduction of
ARS1 origin activity as measured by either plasmid stability or direct chromosomal 2-D origin mapping. Notably
also, MCM, but not ORC, is significantly impaired in its
ability to bind this ARS1 origin DNA in vivo as measured
by ChIP experiments. A simple interpretation of these
observations is that formation of the pre-RC requires both
ORC and a nucleosome positioned by and next to ORC.
Since MCM is also required for the elongation phase of
DNA replication [116], additional cell-cycle restricted
assays for pre-RC assembly are needed to distinguish
whether the primary role of the positioned nucleosome is
in pre-RC assembly during G1 or stability of MCM with
chromatin during S-phase.
If nucleosome positioning can regulate origin activity, a
reasonable prediction is that ATP-dependent chromatin
remodeling machines that move nucleosomes can modulate
the activity of at least some individual origins, particularly
those that might lack the inherent nucleosome positioning
features of ARS1. Given the observations described above,
the ability of these complexes to re-position nucleosomes
either over or around origins could conceivably either
inhibit or enhance origin activity, respectively. To date,
there is a paucity of directed and comprehensive studies
examining the role of chromatin remodeling complexes on
origin activity in yeast. Perhaps this is due to the difficulty
of identifying useful mutant versions of these complexes
(since these complexes also have critical roles in transcription) and/or appropriate origins. However, studies of viral
mediated replication in vitro and in vivo indicate that
chromatin-remodeling complexes can enhance origin activity [117]. And one intriguing and prescient study provides
evidence that the SWI/SNF complex that is required for the
transcription activation of several genes may enhance the
activity of specific origins [118].
In this study, origin activity was assessed in plasmid
stability assays and several different ARSs were examined
in the context of the same plasmid backbone. ARS1 and
ARS307 do not require the SWI/SNF for full plasmid
stability, but ARS121 does, even though ARS1 and
ARS121 confer similar mitotic stabilities to the plasmid
and ARS121 confers greater stability than ARS307. In
mutant strains defective in SWI/SNF, ARS121 plasmid
stability is reduced several fold whereas the stability of
the ARS1 and ARS307 plasmids is unaffected. Interestingly,
an ARS1 mutant plasmid containing a defect in this
origin’s Abf1p binding site requires SWI/SNF for full
stability, suggesting that a sequence-specific replication
enhancer and the SWI/SNF complex play redundant roles
in origin activity. In addition, a mutation in the B1-element
that should reduce ORC’s affinity for ARS1 [23, 24] shows
a similar enhanced dependence on SWI/SNF. These observations are consistent with the proposed role of ORC and
Abf1p in positioning nucleosomes around but outside of
ARS1—when this ability is reduced, ARS1 origin activity
relies more heavily on the SWI/SNF chromatin-remodeling
machine. A critical test of this idea requires nucleosomemapping studies coupled with 2-D origin analysis of
selected natural and mutant origins in wild type and
SWI/SNF defective strains. Additional investigations may
reveal a role for other chromatin remodeling proteins in
origin activity as well.
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3. Roles for ORC in chromatin structure
A role for ORC in establishing domains of specialized
chromatin is now well established [38], and several independent studies indicate that this role is distinct from ORC’s
role in replication initiation at origins [101,119– 123]. A
number of the mechanistic studies relevant to this issue have
used the yeast silent mating-type locus HMRa and some of
the more recent studies will be discussed here.
The elements that nucleate silencing at HMR, the HMR-E
and HMR-I silencers, are closely associated with weak but
nevertheless detectable origin activity, and an early postulate
was that the silencer activity of this element depended on its
origin activity [124]. Obviously, both origin and silencer
activity require binding of ORC to its target site; mutant
silencers or ORC alleles that reduce ORC binding sufficiently reduce both silencer and origin activity at HMRa
[55,120,125]. However, it is clear that ORC’s roles as a
replication and silencing protein are separable. In particular,
it is possible to isolate ORC alleles that show silencing
defects but no discernible replication defects. For example,
the N-terminal f 230 amino acids of the Orc1p subunit
govern a direct and physical interaction between the Sir1p,
one of the four Sirs essential for silencing HMR, and ORC
and yet this N-terminal region is unnecessary for ORC1’s
essential role in replication [123]. Specific alleles of ORC5
can also be isolated that reduce silencing but have no effect
on genome replication [121]. Conversely, some alleles of
ORC2 and ORC5 can provide silencing to HMRa without
providing sufficient replication function to rescue replication defects caused by temperature-sensitive alleles of these
genes [119,121]. Thus, the ORC’s role in silencing, postulated to be recruitment of the Sir1p silencing protein for the
nucleation of silencing [126 – 128], is mechanistically distinct from its replication role at origins.
The multi-subunit heteromeric nature of ORC and its
potential ability to form different conformations and context-dependent protein recognition surfaces probably contribute to its multi-purpose nature in chromosomes. Two
studies indicate that the conformation of yeast ORC can be
regulated. In one, ORC exhibits two distinct conformations
that can be distinguished by electron microscopy [129]. The
presence of origin DNA and ATP stabilizes one conformation. An alternate conformation is observed in the absence
of nucleotide and the presence of single-stranded DNA to
which ORC can bind in a sequence-non-dependent manner.
In another study, the Cdc6p promotes an alternative ORC
conformation in vitro [130]. The mechanistic role for these
ORC conformations is not yet clear. But the existence of
alternate ORC conformations raises the possibility that
subtler conformations of ORC may exist, perhaps dependent
on ORC’s target site and neighboring sequences or proteins,
that could modulate ORC’s roles in chromatin structure or
origin activity.
One recent study indicates that ORC’s target site within a
silencer is quantitatively and perhaps qualitatively different
153
from its target sites in many non-silencer replication origins
[102]. The ORC target site (A/B1-element) within the HMRE silencer binds ORC with about a 10-fold or even higher
affinity than many target sites from active non-silencer
replication origins, including ARS1. Based on estimates of
the cellular ORC concentration [25,131] and comparisons to
other origins, the binding affinity of ORC for its target site
in the HMR-E silencer is greater than necessary for full
occupancy by ORC. Surprisingly, the high-affinity ORC
binding site actually inhibits replication initiation from
HMR in a SIR-independent manner, suggesting that ORC’s
binding to the silencer is optimized for silencer activity and
not compatible with efficient origin activity. Perhaps the
target site for ORC present at the HMR-E silencer promotes
an ORC conformation optimized for interaction with the
Sir1 protein and high-affinity binding is a by-product of that
conformation. Alternatively, extremely stable binding by
ORC may contribute to a stable chromatin structure that
survives the chromosome dynamics of the cell cycle. This
level of binding, well beyond what is necessary for efficient
occupancy by ORC, may prohibit origin firing, perhaps
because it prevents ORC from performing some originspecific role in replication initiation. Many more target sites
must be examined, both in vitro and in vivo, for a clear
picture to emerge. However, these data provide a hint that
differences in the intrinsic nature of ORC – DNA interactions at origins could influence ORC’s role in chromatin
structure.
Other than the silencers, no other ORC binding site in
yeast is known to control specialized chromatin domains,
but three recent studies indicate that ORC has a flexible
ability to adapt new roles in chromatin structure. Two of
these studies focus on an unusual form of SIR-independent
silencing that can be established at HMRa by a dominant
allele of the SUM1 gene, SUM1-1[132,133]. Sum1p normally acts as a direct repressor of meiotic genes [134,135].
Sum1p does not bind to HMRa or function in HMRa
silencing. The SUM1-1 mutant allele, however, has the
amazing capacity to restore silencing in strains lacking
any of the SIR genes, and the Sum1-1p binds to HMRa as
measured by ChIPs. Sum1-1p mediated silencing is similar
to SIR-dependent silencing in that it requires an NADdependent deacetylase, but Sum1-1p uses HST1 instead of
SIR2 for its NAD-dependent deacetylase. Therefore, Sum11p-dependent silencing must be quite different from SIRdependent silencing.
Remarkably, however, Sum1-1p-dependent silencing still
requires ORC; deletion of the Orc1 N-terminus abolishes
Sum1-1p dependent silencing [132]. Sum1-1p’s association
with HMRa requires ORC because mutations in ORC, such
as the orc5-1 allele, reduce a Sum1-1p-HMRa association as
measured by ChIPs [132]. In addition, Sum1-1p interacts
with Orc5p in a two-hybrid assay and with ORC by coimmunoprecipitation [133]. Thus, an alternative form of
ORC-dependent repressive chromatin can be created by a
single amino acid change in the Sum1p. ORC is not required
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for Sum1p’s role in meiotic repression, but even wild-type
Sum1p has some ability to bind ORC [133]. Perhaps ORC
and wild-type Sum1p function together at other as yet
unidentified loci. Regardless, it is striking how little genetic
change is required to create an alternative form of heterochromatin at HMRa that still requires ORC, and intriguingly
the same region(s) of ORC required for SIR-dependent
silencing.
Evidence from the third study suggests that other origins,
not previously known to possess silencer activity, can
provide silencer activity to HMR[136]. Specifically the
2AARS can substitute for the HMR-E silencer and establish
SIR-dependent silencing at HMR. Although 2AARS-dependent silencing requires ORC and the other SIR genes, it is
distinct from HMR-E dependent silencing in that it also
requires another NAD-dependent protein deacetylase SIR2
homolog, HST3 and the Mig1p that has a binding site within
the 2AARS. Thus, an entirely different origin, distinct from
the silencer-associated origin, can establish an ORC-dependent heterochromatin structure. ORC appears readily capable of adapting new roles in chromatin structure by
exploiting certain DNA sequences, chromosomal contexts
and/or neighboring proteins. This capability suggests that
origins may readily acquire alternative non-replication roles
(Section 1.4).
ORC has roles in controlling alternative chromatin structures in Drosophila and possibly other organisms as well.
Perhaps some ORCs mark positions as replication origins
during the rapid divisions of early development in multicellular organisms, and later during development, when the
cell division cycle slows and less active origins are required,
these same chromosomal positions, through their associated
ORCs, may function as specialized chromatin nucleation
sites. Interestingly, studies in both Xenopus and Drosophila
support the idea that the number of active origins is reduced
during development as more defined specialized tissues
begin to form [137,138]. If these inactive origins still bind
ORC, they may serve roles in tissue-specific chromatin
structures and gene expression.
4. Concluding remarks
Many independent studies over the last several years
have established a role for chromosome context and chromatin structure in controlling the efficiency and timing of
origin activation during S-phase in S. cerevisiae. More
recent studies have implicated specialized chromatin-binding proteins, chromatin-modifying enzymes and chromatinremodeling proteins in controlling origin activity. Future
examinations of known and as yet undiscovered chromatin
effects on both global and selective origin regulation will
continue to flourish in S. cerevisiae because of the combination of whole-genome origin maps and the well-recognized experimental advantages offered by this model
organism. The study or metazoan origins and tissue-specific
origin regulation will continue to advance as technologies
for genome-wide studies of replication proteins in chromatin
and replication dynamics improve. It is probable that many
of the rules for chromatin effects on and by origins that we
learn from S. cerevisiae will be applicable at some level to
the tissue-specific regulation of origin activity in metazoans.
Even so, a replication map for even a single mammalian
chromosome that is analogous to the genome-wide replication maps of yeast will provide tremendous fuel for future
mechanistic studies.
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