Identification of Initiation Sites for DNA Replication in the
Human dnmt1 (DNA-methyltransferase) Locus*
Felipe D.
Araujo,
J. David
Knox
,
Shyam
Ramchandani
,
Richard
Pelletier,
Pascal
Bigey
,
Gerald
Price,
Moshe
Szyf
§, and
Maria
Zannis-Hadjopoulos
From the Department of Biochemistry, McGill University and the
Department of Pharmacology and Therapeutics, McGill
University, Montreal, Quebec H3G 1Y6, Canada
 |
ABSTRACT |
Vertebrates have developed multiple mechanisms to
coordinate the replication of epigenetic and genetic information.
Dnmt1 encodes the maintenance enzyme DNA-methyltransferase,
which is responsible for propagating the DNA methylation pattern and
the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5' region of
DNA-methyltransferase 1 (dnmt1) have indicated the presence
of many sequence elements associated with previously characterized
origins of DNA replication. This study tests the hypothesis that the
dnmt1 region containing these elements is an origin of
replication in human cells. First, we demonstrate that a vector
containing this dnmt1 sequence is able to support
autonomous replication when transfected into HeLa cells. Second, using
a gel retardation assay, we show that it contains a site for binding of
origin-rich sequences binding activity, a recently purified replication
protein. Finally, using competitive polymerase chain reaction, we show
that replication initiates in this region in vivo. Based on
these lines of evidence, we propose that initiation sites for DNA
replication are located between the first intron and exon 7 of the
human dnmt1 locus.
 |
INTRODUCTION |
Mammalian DNA replication initiates from multiple sites throughout
S phase (1). These sites are determined both by cis-acting DNA
sequences, known as replicators, and by trans-acting elements, defined
by initiator proteins that bind to the replicator (1, 2). Because of
the complexity of the mammalian genome, initiation of replication has
been studied in greater detail in prokaryotes and lower eukaryotes.
However, a number of techniques, including methods for the isolation of
newly synthesized DNA in combination with competitive PCR techniques,
have led to the identification of new mammalian replication initiation
regions as well as characterization of additional initiation sites at
established origins (3-8). The best example of such a region is the
extensively studied initiation region mapped downstream of the Chinese
hamster dihydrofolate reductase gene (dhfr) (4, 7, 8), where
multiple initiation sites have been shown distributed over a 55-kb region.
Metazoan chromosomal origins of replication comprise specific sequence
motifs. Replication initiation elements can be moved to new chromosomal
sites and still initiate replication (9-11), whereas deletions of
specific sequences in these elements abolish their ability to initiate
replication (11, 12). The ability of specific mammalian DNA sequences
to support autonomous replication of plasmids into which they have been
inserted has been used in the past as an assay for the presence of an
origin of replication in that sequence (13-20). In several instances,
the ability of certain specific sequences to support autonomous
replication of plasmids has been directly validated by mapping of the
same sequences as chromosomal initiation sites for replication in
living cells (16).
Whereas only a few origins of replication have been identified in
mammals, certain sequence motifs have been found to be common in most
of these origins (21). These motifs include A/T-rich regions, ATTA and
ATTTA nuclear matrix attachment motifs, and yeast ARS consensus
sequence elements (WTTTATRTTTW), identified in autonomously replicating
sequences in Saccharomyces cerevisiae (20, 22). Furthermore,
an origin binding activity
(OBA)1 was recently purified
from HeLa cells (23) through its ability to bind to the 186-bp minimal
replication origin of ors 8 (13) and a 36-bp sequence (A3/4)
that is found in a number of mammalian replication origins. OBA
sediments at approximately 150 kDa in a glycerol gradient, and it
cofractionates with DNA polymerases
and
, topoisomerase II, and
replication protein A (23).
In addition to genetic elements, epigenetic components such as DNA
modification by methylation and chromatin structure, have been proposed
to be characteristic determinants of origins of replication (6, 24,
25). We have previously shown that origins of replication like
regulatory regions of genes are differentially methylated (26).
Furthermore, it was recently shown that nonmethylated CpG islands are
enriched in early replicating nascent DNA (6). However, because of the
limited number of mammalian origins of replication that have been
characterized thus far, it is still too early to draw general
conclusions regarding the critical genetic and epigenetic determinants
of origin function and its differential regulation. Additional origins
have to be characterized to allow for understanding of the general
rules governing origin function in mammals.
DNA methylation is a post-replicative covalent modification of DNA that
is catalyzed by the DNA-methyltransferase enzyme (DNMT1) (27-29). In
vertebrates, 60-80% of cytosines in CpG sequences are methylated; the
nonmethylated CpGs are distributed in a nonrandom manner, generating a
pattern of methylation that is gene- and tissue-specific (28). Recent
breakthroughs have provided conclusive molecular evidence for the
hypothesis that DNA methylation of a gene represses its expression (27,
30). DNA methylation can repress gene expression directly by inhibiting
binding of transcription factors to regulatory sequences (30) or
indirectly, by signaling the binding of factors specific for methylated
DNA (31, 32). Two methylated DNA-binding proteins, methyl-CpG-binding proteins MeCP2 and MeCP1 (30, 33), that can repress transcription in a
methylation-dependent manner, have been recently
characterized. The carboxyl-terminal half of methyl-CpG-binding protein
2 contains a repressor domain that associates with the transcription
repressor mSin3A and histone deacetylase (34, 35). Because DNA
replication and methylation are catalyzed by different enzymes, it is
likely that vertebrates have developed multiple mechanisms to
coordinate the replication of the genetic and epigenetic information
(36). In support of this hypothesis, we have shown that the expression of the dnmt1 gene encoding the maintenance DNMT1 enzyme is
regulated with the growth state of cells (37) and that methylation of DNA occurs concurrently with replication (26). Another mechanism that
coordinates methylation of nascent DNA with its replication is the
binding of DNMT1 to proliferating cell nuclear antigen at the
replication fork during the S-phase of the cycle (38). P21, a cyclin
kinase inhibitor that triggers cell arrest, competes with DNMT1 for
proliferating cell nuclear antigen binding and could dislodge it from
the replication fork (38).
We have previously resolved the genomic structure of the gene encoding
the human DNMT1 and showed that it is composed of at least 40 exons and
39 introns spanning a distance of 60 kb (29). While characterizing the
5' regulatory regions of dnmt1, we identified an intronic
sequence immediately upstream of exon 2 that comprised many of the
sequence elements previously described for replication initiation
regions (21). In this study, we tested the hypothesis that this region
of the dnmt1 locus contains initiation sites for DNA
replication, thus physically linking the regulatory region of the gene
encoding the DNMT1 enzyme with an origin of replication in human cells.
Using several lines of evidence based on autonomous replication assays,
OBA binding assays and in vivo mapping by competitive PCR,
we propose that the 5' region of the dnmt1 gene comprises
functional initiation sites for DNA replication.
 |
MATERIALS AND METHODS |
Cell Culture and Plasmid Construction--
HeLa cells
(monolayers) were cultured in Dulbecco's minimal essential medium
supplemented with 10% fetal calf serum. The c1 construct (see Fig.
1A) was generated using genomic subclones of the
dnmt1 pFTR1 that were previously described (29). pFTR1 was
digested with XbaI, and a 2.38-kb fragment-containing
intronic sequence immediately upstream of exon 2 was isolated and
inserted into a XbaI-digested pBluescript SK-plasmid.
DNA Sequencing--
The 2.38-kb insert was fully sequenced by
the dideoxy chain termination method using a T7 DNA sequencing kit
(Amersham Pharmacia Biotech).
Gel Retardation Assay--
The following oligonucleotide
duplexes containing putative OBA binding sites were used: A3/4
oligonucleotides, 5'-CCTCAAGTCTCCAATTTTCCTTTGGCAAATTCCl (sense),
5'-GGAATTTGCCAAAGGAAAATTGGAGACCATTTGAGG (antisense); A3/4 homologous
(dnmt1) oligonucleotides, 5'-TGTTATGGGCTGTTGTCAGACCCAACTGG (sense), 5'-TCCAGTTGGGTCTGACAACAGCCCATAACAA (antisense). The
sense oligonucleotides were radiolabeled in a mixture containing 5 µl of oligonucleotide, 50 µCi of [
-32P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech), and 10 units of T4 polynucleotide
kinase (Boehringer Mannheim) in a final volume of 50 µl and purified
on a Sephadex G25 microcolumn (Amersham Pharmacia Biotech). The
antisense strand was then added (1 µg), and the duplex was annealed
following 5 min of boiling and gradual cooling of the reaction mixture
to room temperature. The DNA binding reaction contained 10 ng of
labeled duplex DNA, 1 µl of poly(dI-dC) (1 µg/µl), 2 µl of 3A
buffer (10 mM Tris-HCl, 80 mM NaCl, 10 mM EDTA, 10 mM
-mercaptoethanol, 1% Triton
X-100, 40% glycerol), 2 µl of 40 nM ATP, and 200 ng of
purified OBA in a 20-µl final volume. In the competition experiments,
10 µg of the respective cold oligonucleotide competitor were added.
The mixtures were incubated for 30 min on ice and then subjected to
electrophoresis on a 4% polyacrylamide gel for 1 h and 30 min at
180 V. The gel was then dried and exposed for autoradiography.
Autonomous Replication Assay--
5 µg of the c1 construct and
pBluescript SK were each separately transfected into HeLa cells by the
calcium phosphate coprecipitation method, and the autonomous
replication assay based on bromodeoxyuridine incorporation and density
shift was performed as described previously (13, 17, 19).
Competitive PCR Assay--
The following primers were used for
the dnmt1 locus (bp 1 was assigned to the first 5' bp
of the c1 construct): c1 region, 5'-AGAGACATCCTGAAGAATGAGTT (p1,
sense, starting at bp 240), 5'-AGTGAGCCGTGATTGCATCA (p2, antisense,
starting at bp 643), 5'-GATTGCATCAGGGGCAGGTCATATAGTTGG (primer used to
design competitor at c1); c2 region, 5'-AGACGTAGAGTTACATCCAG (p3,
sense, ~6.3 kb downstream of p2), 5'-GCTCTTTCAGGTTCTTCTGC (p4,
antisense, ~ 7 kb downstream of p2),
5'-TTACATCCAGAATCAGGAACGCGCACTGAA (primer used to design competitor at
c2); c3 region, 5'-CTACAGGCTCAAGCCACCAT (p5, sense, starting at bp
2906), 5'-TTCCCAAGCTATTCACTAGT (p6, antisense, starting at bp 3300),
5'-ATTCACTAGTCAGGCAATGCTGTCTCAGTC (primer used to design competitor at
c3); c4 region, 5'-CACTCTAGACTGCGGGG (p7, sense, starting at bp 1718),
5'-TCATGCCATTGCACTCTAGC (p8, antisense, starting at bp 2159),
5'-GCACTCTAGCACCACCCAACTATTAGCAGC (primer used to design competitor at
c4); c5 region, 5'-CTCCCGAGTTCAAGCAATTC (p9, sense, ~4 kb upstream of
p1), 5'-GCTGTACAGGGGAAGAGCTG (p10, antisense, ~ 3.5 kb upstream of
p1), 5'-CAAGCAATTCGACTGGGTTTTGCCATGTTG (primer used to design
competitor at c5). Approximate distances between some of the
dnmt1 primers were calculated from Southern blot analysis
(28). The following primers were used for the c-myc locus
(GenBankTM accession number J00120): MO region, 5'-TGCCGTGGAATAACACAAAA
(p11, sense, starting at bp 761), 5'-CTTTCCAGGTCCTCTTTCCC (p12,
antisense, starting at bp 1134), 5'-TAACACAAAAGATCATTTCAGGGAGCAAAC (primer used to design competitor at MO); MF region,
5'-GGTTCTAAGATGCTTCCTGG (p13, sense, starting at bp 7848),
5'-ATGGGTCCAGATTGCTGCTT (p14, antisense, starting at bp 8299),
5'-TGCTTCCTGGGAGAAGGTGAGAGGTAGGCA (primer used to design
competitor at MF). The PCR reactions for c1 and c2 regions were
performed as follows: 3 min at 94 °C, then 30 cycles of 1 min at
94 °C, 1 min at 55 °C, 1 min at 72 °C, followed by 5 min at
72 °C. The PCR reactions for the remaining regions were performed as
follows: 3 min at 94 °C, then 30 cycles of 1 min at 94 °C, 1 min
at 60 °C, 1 min at 72 °C, followed by 5 min at 72 °C.
Isolation of nascent DNA, c-myc PCR primer design, and competitor construction were done as described previously (5, 26, 39,
40).
Bisulfite Mapping Analysis--
The following primers were used
for the dnmt1 site (bp 1 was assigned to the first 5'
bp of the c1 construct): 5'-TTATGTTGTTTAGGGTTGGATT (Morif 1, sense,
starting at bp 6); 5'-TTTATAAGTTATTTTTTTATTATAGTT (Morif 2, sense,
starting at bp 38) 5'-AAAACAACCAACTAATATTCCT (Birom 1, antisense,
starting at bp 415); 5'-TCAAAATAATAACCCAAAACCA (Birom 2, antisense, starting at bp 451). Bisulfite mapping was performed as
described previously (26).
 |
RESULTS |
Sequence Elements Associated with Characterized Origins of DNA
Replication Are Present in the First Intron of the dnmt1
Locus--
Sequence analysis of a 2.38-kb construct (c1 construct)
(Fig. 1, A and
B)-containing intronic sequence immediately upstream of exon
2 revealed several sequence elements that are associated with
characterized origins of replication (21). These elements include a
536-bp stretch that is 77% A/T-rich, at least 19 ATTA and ATTTA
nuclear matrix attachment motifs (21), a perfect match of the 11-bp ARS
consensus sequence (WTTTRTATTTW) of the yeast S. cerevisiae
(22), a methylated CpG cluster (25, 26), and a region homologous to the
binding site of OBA, a human origin binding activity (23).

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Fig. 1.
Sequence elements of the dnmt1
c1 region. A, schematic diagram of the
exon-intron structure of the human dnmt1 locus. Exons are
depicted as vertical bars and numbered above,
introns are depicted as horizontal bars. Regions containing
exons coding for specific functional domains are depicted.
NLS, nuclear localization signal; FTR,
replication foci targeting region; Zn, zinc binding domain;
Sam binding, S-adenosyl methionine binding motif;
Pro-Cys, proline-cysteine catalytic motif; Catalytic domain, region
conserved in cytosine 5'-methyltransferases. ATG, exonal location of
proposed initiation codons. B, sequence analysis of a
2.38-kb construct (c1 construct consists of a XbaI fragment
from ftr 1 phage (28) depicted in Fig. 1A)-containing
intronic sequence immediately upstream of exon 2 exhibited a remarkable
536-bp stretch 77% A/T rich. At least 19 ATTA and ATTTA nuclear matrix
attachment motifs, as well as a perfect match of the 11 bp ARS
consensus sequence (WTTTRTATTTW) reside in this region. A methylated
CpG cluster and A3/4 homologous region are also present.
Asterisk (*), CpG methylation observed in 50% of the clones
tested.
|
|
The c1 Construct Is Able to Support Autonomous Replication--
To
ascertain whether the dnmt1 sequence indeed contained an
origin of replication, we first tested the ability of the c1 construct to support autonomous replication using a density shift assay following
bromodeoxyuridine incorporation into nascent DNA, as described
previously (13, 17, 19). The results (Fig.
2A) demonstrate a density
shift of the c1 construct corresponding to incorporation of
bromodeoxyuridine into one (heavy-light) or both of the nascent DNA
strands (heavy-heavy), indicating one and two or more rounds of
replication, respectively. The SK plasmid vector, on the other hand,
did not exhibit a similar shift, and all plasmid DNA was recovered in
the unreplicated (light-light) form of input DNA (Fig. 2B).
The linearity of each gradient was verified by measuring the refractive
index of every other fraction.

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Fig. 2.
c1-bearing plasmid (A) and a
pBluescript SK (B) control were transfected into HeLa
cells, and a density shift assay following bromodeoxyuridine
incorporation was performed as described previously (19). The
different fractions were spotted onto a Hybond N+ membrane
and hybridized with a 32P-labeled probe bearing the vector
sequence, and the intensity of the signal at each fraction (lower
panels) was quantified by PhosphorImaging and presented as
relative DNA content (signal at specific spot/total signal). The
linearity of each gradient was verified by measuring the refractive
index (R. I.) of every other fraction and plotted as a
linear graph.
|
|
The dnmt1 A3/4 Homologous Region Is Able to Bind OBA--
Sequence
analysis revealed a 30-bp stretch that is 86% homologous to the 36 bp
A3/4 sequence, identified as part of the binding site for
OBA.2 An oligonucleotide of
this 30-bp dnmt1 sequence was synthesized, and its binding
activity to OBA was assayed by gel retardation in the presence of
excess nonspecific competitor poly(dI-dC). The results (Fig.
3) show that the dnmt1
oligonucleotide is able to bind to and form a complex with OBA
(lane 3) of the same size as the complex formed when the 36 bp A3/4 oligonucleotide is used (lane 6). This binding is
specific, because the dnmt1 oligonucleotide was able to
compete for the OBA binding (lanes 4 and 8) as
effectively as the 36-bp A3/4 oligonucleotide when it was used as
competitor (lanes 5 and 7).

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Fig. 3.
, Sequence analysis of the first intron of the
dnmt1 exposed a 30-bp stretch 86% homologous to the
36-bp A3/4 sequence. A 32P-labeled double-stranded
oligonucleotide bearing the 30-bp dnmt1 sequence
(Met.) or the consensus A3/4 sequence was incubated with 200 ng of purified OBA in the presence excess nonspecific competitor
poly(dI-dC) (1 µg). Specific competitors were added (10 µg of cold
Met. or A3/4 oligonucleotide) in the competition experiments.
|
|
Initiation Sites for DNA Replication Are Present within the dnmt1
Locus--
To quantitatively analyze the replication origin activity
of the c1 construct and map the initiation site(s) in it, a series of
competitive PCR experiments were performed to measure and compare nascent DNA abundance of five different regions within the
dnmt1 locus (Fig. 4). The top
gel (Fig. 4AI) of each of the regions (c1-c5, MO, and MF)
corresponds to the competitive PCR amplification of HeLa genomic DNA
used to standardize the differences among primers and competitors with
respect to their amplification efficiencies. The linearity of each of
the competitive PCR analysis was verified by plotting the ratio of
competitor DNA concentration over target DNA concentration
(y axis) versus the concentration of competitor DNA (x axis) (Fig. 4B). Small nascent DNA ranging
in size between 800 bp to 1.3 kb was isolated by the nascent strand
extrusion method (5, 26, 40) followed by sucrose gradient sedimentation and further purification on agarose gel, as described previously (5).
The bottom gel (Fig. 4AII) of each of the regions (c1-c5, MO, and MF) corresponds to the competitive PCR amplification of HeLa
nascent DNA, and the bar graphs of the c-myc
locus (Fig. 4C) and the dnmt1 locus (Fig.
4D) represent the nascent DNA abundance obtained by the
respective competitive PCR assays. Two regions, MO and MF, within the
well characterized initiation region associated with the
c-myc gene (39) were used as control for the quality of the
nascent DNA preparation (Fig. 4C). The results (Fig.
4C) show that a previously characterized c-myc
origin of replication (MO) is present in the nascent DNA preparation at
6 × 10 3 copies of nascent DNA, whereas no signal is
observed for a region located 7 kb downstream from the origin of
replication (MF), indicating that the nascent DNA used did not contain
degraded parental DNA (5, 26). The same preparation of nascent DNA was
used to determine the relative abundance of five regions within the
dnmt1 locus. The results (Fig. 4D) indicate that
two regions of DNA located between the first intron and the seventh
exon of dnmt1 (c1 and c3) are highly abundant in the nascent
DNA fraction, containing approximately 3 × 10 4 copies of nascent DNA each. In contrast, the relative abundance of
regions of dnmt1 residing approximately 3 kb upstream of the c1 region (c5) or 5 kb downstream of the c3 region (c2) are far less
abundant, containing 6 × 10 3 copies of nascent DNA
(c5) and no nascent DNA (c2), respectively. The data are consistent
with the hypothesis that at least two major initiation sites for DNA
replication are localized between the first intron and exon 7 of the
dnmt1 gene.

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Fig. 4.
Competitive PCR mapping of an in
vivo origin of replication residing in the dnmt1
locus. A series of competitive PCR experiments were
performed to measure and compare the abundance of 5 different regions
within the dnmt1 locus (c1-c5) in nascent DNA. To normalize
the differences in primer and competitor amplification efficiencies,
HeLa genomic DNA was used as template for the competitive PCR assay
(AI, top gel for each indicated regions). The
linearity of each of the competitive PCR analysis was verified by
plotting the ratio of competitor DNA concentration over target DNA
concentration (y axis) versus the concentration
of competitor DNA (x axis) (Fig. 4B). Small
nascent DNA ranging in size between 800 bp to 1.3 kb was used as
template for the competitive PCR assay (4AII, bottom
gel for each of the indicated regions). The competitor molecule
number used in each competitive PCR is displayed on top of every gel.
As a control for the purity of the nascent DNA preparation, we
determined the abundance of the c-myc origin of replication
(MO) and a sequence residing 7 kb downstream (MF) (C). The
position of each of the tested regions is illustrated in the schematic
diagrams of both dnmt1 and the c-myc locus below
the respective bar graphs. The bar graph results
(D) represent nascent DNA abundance for each indicated
dnmt1 region. ATG, exonal location of proposed initiation
codons; NLS, nuclear localization signal.
|
|
A Methylated CpG Cluster Is Associated with the Initiation Sites
for DNA Replication--
A number of origins of replication, among
them ori
at the dhfr locus, have been
previously shown to be associated with a methylated CpG cluster (25),
whereas other origins of replication have been shown to be unmethylated
in the associated CpG sequences (6, 26), suggesting differential
methylation of replication origins. To determine whether
dnmt1 is associated with either a methylated or unmethylated
CpG cluster, we performed a bisulfite methylation mapping analysis of a
CpG cluster located within the c1 construct region (Fig.
5A). Twelve clones were
selected, and the eight CpGs residing in this region were analyzed in
each clone with respect to their methylation status. Six of them were
methylated in all clones tested; 1 CpG was unmethylated, and
interestingly, 1 CpG was methylated in 6 of the 12 clones tested (Fig.
5, A and B). A sample result (Fig. 5B)
of the bisulfite analysis indicates the methylated CpGs and their
respective positions. The results show that the dnmt1
initiation region is associated with a methylated CpG cluster, as was
previously demonstrated for the dhfr ori
(25).

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Fig. 5.
A methylated CpG cluster is associated with
the DNMT1 replication initiation region. A, bisulfite
mapping analysis of the state of methylation of a CpG cluster located
upstream of the dnmt1 A/T-rich region (Fig. 1B).
The physical map of the c1 region exhibits the position of the 8 CpGs.
DNA prepared from HeLa cells was subjected to bisulfite treatment, and
the primers indicated under "Materials and Methods" were used to
amplify the CpG cluster-containing region. The amplified fragments were
subcloned, and 12 clones were sequenced. The state of methylation of
each CpG is indicated in the table (A). Methylated CpGs are
indicated by a black ball and stick, partially methylated
CpGs are indicated by a checkered ball and stick, and
unmethylated CG is indicated by a white ball and
stick. B, a gel sample of the bisulfite analysis is
shown; the positions of the methylated CpGs is indicated by the
numbered balls and sticks.
|
|
 |
DISCUSSION |
This paper describes the identification of initiation sites for
DNA replication located between the first intron and exon 7 of the
dnmt1 locus. This finding is supported by several lines of
evidence. First, a plasmid containing the c1 region of dnmt1 is able to replicate autonomously. Second, this region contains a
binding recognition sequence and binds to OBA, a protein that binds to
origins of replication. Third, this region of dnmt1 contains several sequence elements characteristic of known origins of DNA replication, such as, an A/T-rich region, matrix attachment motifs, a
methylated CpG cluster, and a perfect match to the yeast ARS consensus
sequence (21). Finally, this region is abundant in nascent DNA, as are
other characterized origins of replication, as determined by
competitive PCR. Two primary replication initiation sites were
identified, one localized in the c1 region and another localized in the
c3 region; these regions are approximately 2 kb apart from each other
(Fig. 4D). This finding is similar to recently described
observations in the Chinese hamster ovary dhfr origin of
replication, where initiation was shown to occur primarily from two
sites, ori
and ori
' (7).
The data presented in this paper further support the previous
observations that regions of initiation of DNA replication share common
sequence features (21). A 536-bp, 77% A/T-rich element is present in
the c1 region of the dnmt1 locus, suggesting a possible function as a low melting region or as a DNA-unwinding element (21).
This region also contains a perfect match for the yeast ARS consensus
sequence, although previous reports have indicated that these sequences
are not essential for mammalian origin function (18, 21). Nineteen ATTA
and ATTTA matrix attachment motifs are present in the 2.4-kb c1
construct; these motifs constitute the core elements recognized by the
homeobox domain from species as divergent as flies and humans and are
frequently present in matrix attachment sites of several eukaryotic
genes in addition to numerous eukaryotic and viral origins of DNA
replication (41).
A methylated CpG cluster is associated with the dnmt1
replication initiation region. DNA methylation is now recognized as a
fundamental mechanism of epigenetic regulation of genomic processes such as transcription, recombination, imprinting, development, carcinogenesis, and replication timing (36). Some origins of replication bear a cluster of heavily methylated CpG sites (25), whereas other origins, such as c-myc, are associated with a
nonmethylated CpG cluster (26). Taking these findings into account, it
seems that origins of replication exhibit differential methylation
patterns similar to those observed in promoter elements of several
genes (36). It remains unclear what role methylation plays in
replication. It has been recently suggested that early activated
origins are associated with nonmethylated CpG islands (6). The
identification of origins that are representative of either methylation
profile will allow the testing of the hypothesis that methylation plays a role in differential activation of origins of replication.
The c1 construct containing all the structural elements mentioned above
is able to support episomal autonomous replication when transfected
into HeLa cells. Results from utilization of in vivo assays
of autonomous replication have been controversial, because some studies
have demonstrated that only large fragments (>10 kb) could support
autonomous replication (15), whereas several other studies showed that
small specific sequences could successfully support autonomous
replication (13-19). In addition, plasmids carrying ori
of the dhfr locus replicated autonomously both in
vivo and in vitro regardless of their size, whereas
plasmids of equivalent size inserts, but with random sequence, did not (16). Furthermore, the capacity of several specific sequences to
support autonomous replication of plasmids correlates directly with
their chromosomal mapping sites (16).
Regarding the significance of the co-localization of initiation of
replication with the regulatory region of the dnmt1 gene, an
attractive possibility would be that the physical association of the
dnmt1 regulatory region with initiation sites for DNA
replication plays a role in coordinating the replication of the genetic
information with that of the epigenetic information. Future experiments
should resolve this question.
In summary, several lines of evidence, both structural and functional,
demonstrate the presence of at least two major initiation sites for DNA
replication residing in the region contained between the first intron
and exon 7 of the dnmt1 locus.
 |
FOOTNOTES |
*
This work was supported by a grant from the Medical Research
Council of Canada (to M. S. and M. Z.-H.) and a grant from Cancer Research Society (to G. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF119248.
§
To whom correspondence should be addressed: Dept. of Pharmacology
and Therapeutics, McGill University, 3655 Drummond St., Montreal,
Quebec H3G 1Y6, Canada. Tel.: 514-398-7107; Fax: 514-398-6690; E-mail:
mszyf{at}pharma.mcgill.ca.
2
M. Ruiz, D. Matheos, G. B. Price, and M. Zannis-Hadjopoulos, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
dhfr, dihydrofolate reductase;
kb, kilobase(s);
bp, base pair(s);
dnmt1-DNA-methyltransferase 1, ARS, autonomous replicating
sequence;
OBA, origin-rich sequences binding activity;
PCR, polymerase
chain reaction;
DNMT1, DNA-methyltransferase 1.
 |
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