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(Received for publication, July 27, 1995; and in revised form, November 4, 1995) From the
To study the organization of DNA replication in mammalian rRNA
genes, the sites of initiation of DNA synthesis in rat and human rRNA
genes were mapped by two independent techniques. In rat cells the
growth of the nascent DNA chains was blocked by Trioxsalen cross-links
introduced in vivo. The fraction of ``restricted''
nascent DNA chains labeled in vivo was isolated, and the
abundance in this fraction of cloned ribosomal DNA sequences was
determined by hybridization. In the experiments with human cells, the
nascent DNA chains were allowed to grow unrestricted for a certain
period of time and the movement of the replication forks along the rRNA
genes was followed by hybridization of cloned ribosomal DNA sequences
to the ``unrestricted'' nascent DNA fragments fractionated
according to size. The results show that in both rRNA genes there are
two well defined regions of initiation of DNA synthesis. The first one
is located upstream of the transcription units and the second one is
located at the 3`-end of the coding regions of the ribosomal DNA
repeats.
A number of techniques for mapping sites of initiation of DNA
synthesis in vivo have been developed during several recent
years. However, their application has led to conflicting results as to
whether origins of DNA replication are located at defined positions in
mammalian genomes. In the cases when methods that analyze the newly
synthesized DNA chains were applied to the dihydrofolate reductase
(DHFR) ( Since the problem of the
organization of initiation of DNA replication in the rRNA gene cluster
or elsewhere in the mammalian genome has not been satisfactorily solved
so far, in the present article we used two well established biochemical
procedures developed by Anachkova and Hamlin(7) , and by
Vassilev and Johnson(8) , to map replication origins in rat and
human rRNA genes, respectively. We modified these procedures to avoid
certain drawbacks of the original protocols and the results we obtained
did not support the hypothesis of random initiation of DNA replication in vivo. We came to the conclusion that in both rat and human
rRNA genes, DNA replication most probably initiates at two well defined
areas located a few kilobases upstream of the promoter and at the
3`-end of the transcription unit, and that no initiation of DNA
synthesis normally occurs outside these zones.
Figure 3:
Physical map of the rat rDNA repeat and
DNA probes. Indicated are the positions of the transcription unit (heavy line) and of 18 S and 28 S RNAs (filled
boxes). The location of the probes used for hybridization with the
nascent DNA fragments synthesized between the Trioxsalen cross-links
are shown below the map. The following abbreviations were used: E, EcoRI; EV, EcoRV; S, SauI.
Figure 5:
Mapping the replication origins in the
human ribosomal DNA repeat. The diagram represents the physical map of
the human rDNA repeat. The positions of the transcription unit (heavy line) and of 18 S and 28 S RNA (filled boxes)
are indicated. EcoRI restriction fragments A (7 kb), B (6 kb),
C (11 kb), and D (19 kb), and the positions of the five rDNA probes are
shown under the map. The 1.5-, 4-, 8-, and 15-kb nascent DNA fragments
are schematically represented by horizontal lines and are
arranged on top of the physical map of human rDNA repeat. To satisfy
the hybridization results presented in Table 1these fragments
should have initiated within two different initiation zones located 5`
of the transcription unit and at the 3`-end of the transcription unit (open boxes), respectively. For comparison in the figure are
included the estimated positions of the human rDNA replication origins
obtained in other laboratories (bottom).
Figure 1:
Diagram of the restricted nascent
chains growth experimental approach. Following Trioxsalen
cross-linking, cells were allowed to synthesize DNA in the presence of
BrdUrd and [
The
unrestricted nascent chain growth technique was developed by Vassilev
and Johnson (8) and was used to map ori-
Figure 2:
Diagram of the unrestricted nascent chains
growth experimental approach. Following pulse labeling with BrdUrd and
[
We have
modified the original protocol of Vassilev and Johnson (8) in a
number of ways. The method has been introduced for detection of origins
of replication in single copy sequences and that is why the selected
unique DNA probes were amplified by the polymerase chain reaction.
Since the rRNA gene family occurs as naturally multicopy, this step was
omitted in our protocol. The most important modification was that we
used the size-fractionated nascent DNA fragments labeled in
vivo, rather then labeled in vitro. For this reason the
label was
Figure 4:
Hybridization of rat ribosomal DNA clones (A) and of the fragments of clone Rr56 after digestion with EcoRI, EcoRV, and SauI (B) with in vivo labeled nascent DNA fraction synthesized between the
Trioxsalen cross-links. 1 µg of the DNA probes were dot-blotted on
nitrocellulose filters and hybridized with 2
To map the sites of initiation of DNA synthesis in the rat
and human ribosomal DNA repeats, we applied two biochemical approaches,
proven to be adequate for localization of mammalian origins of
replication. They have been tested on the yeast ARS1 (14) and
the SV40 (8, 24) origins of replication as model
systems, and were applied to a number of other mammalian replication
origins(7, 14, 19, 20, 21, 22, 23) .
The positions determined by these approaches excellently concur with
the positions determined by other methods(1) . The application
of these two techniques to the rat and human rRNA genes enabled us to
map a well defined zone of initiation of bidirectional DNA replication
a few kilobases upstream of the coding regions, and also a less well
expressed zone of initiation near the 3`-end of the transcribed units.
These results are fully consistent with the findings of Coffman et
al.(5) , that a 1.38-kb sequence, located immediately
upstream of the promoter, and to a lesser extent a 7-kb sequence,
located at the 3`-end of the 28 S coding region of human ribosomal DNA,
serve as efficient substrates in an in vitro replication
system involving proteins from human cells (Fig. 5). The
location of the upstream initiation zone agrees with the major
initiation sites obtained with two-dimensional gel electrophoresis (3) and nascent strand abundance analysis of human rRNA genes (4) (Fig. 5). However, the conclusions that lower
frequency initiation sites are distributed throughout most of the
nontranscribed spacer (3) and the coding region (4) are not consistent with our results. The reasons for this
discrepancy are not clear at present. The two-dimensional gel
electrophoresis is the technique of choice for mapping origins of
replication in genomes with low complexity and genomes that do not take
up DNA precursors readily, since the method does not involve labeling
of nascent DNA. When applied to mammalian genomes, it relies on a
number of assumptions that are not proven to be always
valid(25) . For instance, the method involves enrichment for
replication bubbles by using their presumed association with the
nuclear matrix and assumes that no structural changes or nicks will
occur in DNA during the isolation procedure. The reason the nascent
strand abundance technique leads to the conclusion that initiation can
occur throughout the ribosomal DNA repeat unit could be that the
nascent DNA fractions were not well purified from genomic DNA, or that
DNA have been fragmented in the course of the purification procedure.
In this way random genomic DNA fragments will be present in the nascent
DNA fraction, or fragments from longer nascent chains will be present
in the population of shorter chains, both cases leading to a certain
randomization of the results. In our experiments the careful
purification of the nascent fragments from random genomic DNA was
critical for obtaining meaningful results. For this reason we isolated
the nascent DNA fragments by immunoprecipitation. In this way we
avoided the risk of compromising the results by using nascent DNA
fractions not purified from genomic DNA, because immunochemical
specificity can be considered almost absolute. In addition, we have
followed the hybridization signal of the in vivo labeled
nascent DNA fragments, thus eliminating the effect of any incidentally
present nonlabeled contaminating DNA. Finally, in our experiments the
fractionation of the newly synthesized DNA was done at the very first
step of the isolation procedure and thus any possible artifacts from
DNA degradation were avoided. A possible source for errors in the
experiments with the human rRNA genes could be the use of a single
nucleoside precursor to label DNA since the G + C content of
probes 1 (75%) and 2 (71%) was higher than that of probes 3, 4, and 5
(51, 47, and 56%, respectively). However, this fact should not affect
our results since we deduce the positions of the replication origins
from the length of the nascent fragments that hybridize with the
different probes, rather than from the strength of the hybridization
signal itself. Nevertheless, to make sure that our conclusions have not
been biased because of the different G + C content of the probes,
the same experiments were performed with a different precursor,
[ A support for our conclusion
that there are specific replication origins in the ribosomal DNA repeat
came from the primary structures of the predicted origin regions.
Analyses of the primary structure of other known mammalian chromosomal
origin regions have revealed the existence of certain sequence and
structural elements that are found in zones of initiation of DNA
replication more frequently than would be expected by
chance(26, 27, 28) . These include A +
T-rich tracts, sequences similar to the yeast ARS and sequences similar
to Drosophila SAR, transcription factor binding sites, and DNA
unwinding elements. These sequence elements are found at origins of
replication in simple eukaryotic genomes such as yeast and animal
viruses and their role in initiation of DNA replication has been
determined by biochemical and functional assays. The primary structures
of the upstream initiation zones of both rat and human ribosomal DNAs
have been published and we searched a 2.52-kb EcoRI/SauI rat nontranscribed spacer sequence (29) and a 4.58-kb BamHI/EcoRI human
nontranscribed spacer sequence (30) for such common features (Fig. 6). The numberings of the nucleotides given hereafter are
according to Financhek et al.(29) and Sylvester et al.(30) , respectively. Both sequences exhibit A
+ T-rich tracts that contain yeast ARS-like sequences (31) and Drosophila SAR sequences(32) . In the
rat origin region there is a cluster of three SAR sequences starting at
nt position 198, one at 862, and one at 1943. In the human replication
initiation zone two SAR sites occur at nt positions -4830 and
-1780 and three start at nt position -4290. Two sequences
similar to yeast ARS at nt positions 1624 and 2106 are found upstream
of the rat rRNA genes, and the human initiation zone contains four
ARS-like sequences at nt positions -3785, -3764,
-1374, and -1708, respectively. The initiation zones
contain potential binding sites for at least two proliferation-specific
transcription factors that may function in initiation of DNA
replication, Oct-1/NFIII, and p53. The rat initiation zone contains a
9/10 match to the consensus binding site of Oct-1/NFIII (36) ,
while the human one contains two perfect matches at nt positions
-3968 and -3962. A putative binding site for the protein
p53 (37) is located at nt position 1041 in the rat DNA and at
-4509 in the human DNA. In addition, the sequences contain
extensive polypyrimidine tracts, which are identified as
sequence-specific start sites for the DNA polymerase-primase complex in vitro(38) .
Figure 6:
Organization of common modular sequence
elements in the predicted 5`-initiation zones of rat and human
ribosomal DNA repeats. The initiation regions are diagramed
schematically, with nucleotide positions according to Financhek et
al.(29) and Sylvester et al.(30) . The
sequence elements are described in the text. The following symbols were
used: filled triangle, SAR; open triangle, ARS; filled box, pyrimidine tracts; open box, DNA
unwinding elements.
The analysis of the organization of
origins of replication in Escherichia coli, yeast, and SV40
has shown that the minimal essential cis-acting sequence
required to initiate DNA replication contains a genetic component that
is easily unwound(33, 34) . The DNA unwinding elements
are determined by base stacking interactions between nearest-neighbor
dinucleotides and is not simply a function of A + T content, but
depends on the specific DNA sequence (34) . We used the
computer program Thermodyn (35) to perform a sliding window
analysis of the helical stability ( The existence of these
origin-related sequence elements in the predicted zones of initiation
of DNA synthesis strongly supports the conclusion that they function as
replication origins in vivo. These data are consistent with
the hypothesis that in eukaryotes replication initiates within clusters
of redundant modular elements associated with DNA unwinding
function(39) . We propose that eukaryotic cells have evolved a
limited number of short sequence elements that are used as modules to
build different control regions, including regions of low helical
stability that can serve as origins of DNA replication. Further studies
are necessary to elucidate whether all, or only a subset of these
sites, are used as origins and how, in the course of development and
differentiation, chromatin is organized in such a way that some of the
potential initiation sites are blocked, while others are made
accessible for the assembly of the replication initiation complex.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2608-2614
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)gene domain in Chinese hamster ovary cells, which
is the most studied model for initiation of mammalian DNA replication,
the investigators were able to determine the presence of well defined
origins of bidirectional replication(1) . On the other hand,
the analysis of genomic DNA for the presence of replication bubbles and
forks by the method of two-dimensional gel electrophoresis failed to
indicate the presence of such origins(2) . The rRNA genes are
repeated tandemly about 400 times in mammalian genomes. This makes them
a suitable model for studies of initiation of DNA replication without
the need for synchronization of the cells and amplification of the
nascent DNA fragments. Three recent papers have analyzed the initiation
of DNA replication in the rRNA gene cluster of human cells. In the
first one, the authors used two-dimensional gel electrophoresis and
concluded that initiation of replication takes place throughout most of
the nontranscribed spacer, but not in the transcription unit and the
adjacent regulatory elements(3) . In the second paper, the
authors used a method called ``nascent strand abundance
analysis,'' based on a combination of sedimentation and
electrophoretic fractionation of DNA. They reached the conclusion that
although initiation of DNA replication was more frequent a few kilobase
pairs upstream of the transcribed region, it could occur everywhere in
the ribosomal DNA repeat, including the transcription unit
itself(4) . In the third paper, the authors studied the in
vitro replication of plasmids containing cloned human ribosomal
DNA sequences and showed that replication initiated specifically within
two 7-kb DNA fragments located upstream of the promoter and downstream
of the 3`-end of the coding region(5) . The results of the
first two papers imply that initiation of DNA synthesis may be a random
process. However, there is evidence indicating that initiation of DNA
replication follows a well defined pattern in vivo. This
pattern is specific for each cell line and varies during embryogenesis
and development. Thus, certain genes or sequences are replicated early
in S-phase in some cells, while the same genes and sequences are
replicated late in the S-phase of other cells(6) . A second
argument against the concept of random initiation of DNA replication is
that a completely random initiation, regardless of the number of
initiation events and the length of the S-phase, would leave a part of
DNA unreplicated in each cell cycle.
Cell Cultures
Guerin ascites tumor cells were
propagated in Swiss male albino rats. At days 7-9 after
inoculation (late log phase), the ascites liquid was withdrawn under
sterile conditions and the cells were further cultured in minimal
essential medium for suspension cultures (Sigma), buffered with 50
mM HEPES. HeLa cells were grown as suspension cultures in
minimal essential medium, supplemented with 5% fetal bovine serum
(Sigma). Genomic DNA was randomly labeled with 0.025 µCi/ml
[
C]dT (DuPont, 50 mCi/mmol) for 24 h.Isolation of ``Restricted'' Nascent DNA
Fragments
Cells were spun down at 800 
g and
resuspended in fresh medium without serum to make 5
10
-10
cells/ml. DNA was cross-linked by four
successive treatments with Trioxsalen and near UV light as described
previously (9) to give one Trioxsalen bridge per 1.5 kb on the
average. Cross-linked cells were incubated in the presence of 50
µM BrdUrd (Sigma) and 20 µCi/ml
[
H]dT (70-90 Ci/mmol), or
[H]dC (20-40 Ci/mmol, DuPont) for 1 h to
label the nascent DNA fragments synthesized between cross-links. Cells
were lysed in 0.5% SDS, 1 M NaCl, 10 mM EDTA, 50
mM Tris-HCl, pH 8, and the proteins were digested with 200
µg/ml Proteinase K (Merck) at 37 °C for 4 h. After
deproteinization with phenol/chloroform (1:1) and with chloroform, 1
volume of ethanol was overlayered and the high molecular weight
chromosomal DNA was recovered by spooling on a glass rod. DNA was
dissolved in 10 mM Tris-HCl, 1 mM EDTA, pH 7.4, to
give 200-500 µg/ml, made 0.2 M in NaOH by adding 1 M NaOH, and centrifuged in 5-20% sucrose density
gradients prepared in 0.2 M NaOH, 1 mM EDTA in
Beckman SW 27 rotor at 25,000 rpm, 10 °C, for 18 h. Aliquots were
counted, the fractions containing the nascent DNA chains were pooled
together, and DNA was immunoprecipitated as described later in the
text.Isolation of ``Unrestricted'' Nascent DNA
Fractions
Exponentially growing HeLa cells were spun down and
resuspended in fresh medium without serum to make 5
10-10 cells/ml. They were incubated with 50
µM BrdUrd and 10 µCi/ml [H]dC,
or [H]dT for 10 min. Labeling was terminated by
pouring the cell suspension in 10 volumes of ice-cold 0.14 M NaCl, 0.01 M phosphate buffer, pH 7, and DNA was isolated
as in the previous paragraph. To size fractionate nascent DNA chains,
DNA was dissolved in 10 mM Tris-HCl, 1 mM EDTA, pH
7.4, to make approximately 500 µg/ml, made 0.2 M in NaOH
and applied on top of linear 5-20% sucrose density gradients
prepared in 0.2 M NaOH, 1 mM EDTA. Gradients were
centrifuged in Beckman SW 27 rotor at 20,000 rpm at 10 °C for 18 h.
Tubes were unloaded from the bottom and the respective size fractions
of DNA were pooled together and recentrifuged under the same
experimental conditions using Beckman SW 41 rotor.Immunoprecipitation of the Nascent DNA Strands
The
nascent DNA fractions recovered from the gradients were dissolved in
0.14 M NaCl, 0.01 M phosphate buffer, pH 7,
containing 0.5% Tween 20 and 100 µg/ml bovine serum albumin in a
final volume of 400 µl. An equal volume of monoclonal anti-BrdUrd
antibody (Beckton and Dickinson) was added, and after 1 h at room
temperature the antigen-antibody complex was precipitated with an
excess of second antibody (anti-mouse IgG rabbit IgG fraction, Sigma).
After another hour at room temperature the samples were kept at 4
°C overnight and the precipitate was collected by centrifugation in
an Eppendorf microcentrifuge for 10 min. It was washed with 0.14 M NaCl, 0.01 M phosphate buffer, pH 7, resuspended in 200
µl of 1 mM NaCl, 0.5% SDS, 50 mM Tris-HCl, 10
mM EDTA, pH 7, and digested with 200 µg/ml Proteinase K
(Merck). After deproteinization with phenol/chloroform (1:1) and
chloroform, DNA was precipitated with 2.5 volumes of ethanol.Hybridization and DNA Probes
For dot-blot
hybridization, DNA was loaded onto nitrocellulose membranes (Hybond-C,
Amersham) as recommended by the manufacturer using a manifold
dot-blotter (Bio-Rad). Hybridization was carried out under stringent
conditions (7% SDS, 0.25 M phosphate buffer, 1% bovine serum
albumin, at 68 °C overnight). The membranes were rinsed with 0.3 M NaCl, 0.03 M sodium citrate, pH 7, at room
temperature (twice), washed with 0.3 M NaCl, 0.03 M sodium citrate, pH 7, 0.1% SDS at 68 °C for 30 min (twice),
and finally rinsed with 0.015 M NaCl, 0.0015 M sodium
citrate, pH 7, at room temperature. Areas of the membrane containing
individual dots were cut out and counted in Beckman liquid
scintillation counter LS1800. The rat ribosomal DNA probes (10, 11, 12) were as follows: RrII was a
12.6-kb EcoRI/EcoRI fragment; RrIV was a 13-kb EcoRI/EcoRI fragment; Rr101 was a 1.9-kb EcoRI/EcoRI fragment; Rr56, a 5.2-kb EcoRI/HindIII fragment; Rr133, a 5.9-kb HindIII/EcoRI fragment; Rr161, a 6.9-kb EcoRI/EcoRI fragment; and Rr151, a 3.9-kb EcoRI/EcoRI fragment (see Fig. 3for probe
locations). The human ribosomal probes were kindly provided by J.
Sylvester: probe 1 was B, a 1.2-kb EcoRI/SalI fragment; probe 2, D
, a
0.96-kb EcoRI/SalI fragment; probe 3, D
,
a 0.3-kb XbaI/XbaI fragment; probe 4, C
,
a 0.4-kb EcoRI/BamHI fragment; and probe 5,
C
, a 0.47-kb HindIII/BamHI fragment (see Fig. 5for probe locations). 1.2-kb DNA fragment excised from
plasmid pLTRdhfr26 (ATCC) and containing the mouse DHFR gene, and a
211-base pair DNA fragment excised from plasmid pH
A-IVS-I (13) and containing the 5`-end of human -actin gene, were
used as controls.
Experimental Approach
The restricted nascent
chain growth technique was originally developed in our
laboratory(9) , and has been successfully used to map
``ori-'' in the DHFR domain of Chinese hamster
ovary cells (7) and to isolate a number of mouse replication
origins(14) . The rationale behind this approach is depicted
schematically in Fig. 1. Cells whose DNA had been uniformly
labeled with [C]dT were treated with Trioxsalen
and long wave ultraviolet light to introduce cross-links in DNA in
vivo and were incubated with BrdUrd and
[H]dC, or [H]dT for 1 h. In
the course of the incubation BrdUrd and the radioactive precursors are
incorporated into three classes of newly synthesized DNA. (i) As a
result of excision repair short stretches of labeled DNA are
synthesized(15) , which are covalently attached by both their
ends to the high molecular weight DNA and during all subsequent
procedures remain bound to it(16, 17) . (ii)
Elongation of the already initiated DNA chains continues until the
replication forks reach Trioxsalen cross-links, where they will stall.
This produces labeled DNA stretches covalently linked to the high
molecular weight DNA by their 5`-ends. This process will take place
only during the first 2-3 min of the labeling period, since the
distances the replication forks have to travel to reach the first
cross-link are a few kilobases at the most. (iii) Short DNA sequences
initiated at origins of replication, located between Trioxsalen
cross-links, are synthesized. They are not ligated to the high
molecular weight DNA and under denaturing conditions are released from
it to form a fraction of newly synthesized fragments. Upon
centrifugation in alkaline sucrose density gradients, cross-linked
genomic DNA sediments to the bottom, while the fragments synthesized at
origins of replication sediment as a well defined peak in the upper
half of the gradient(16, 17) . The material of this
peak was collected and further purified by immunoprecipitation with
anti-BrdUrd antibody. The course of purification of the nascent DNA
chains was monitored by measuring the H/C
ratio and the specific radioactivity of DNA. After the
immunoprecipitation, the specific radioactivity of the nascent DNA was
equal to that of control genomic DNA, uniformly labeled under the same
conditions. This was an indication that the purification procedure had
efficiently eliminated any contaminating genomic DNA. The purified
labeled in vivo nascent DNA was hybridized to dot-blotted
cloned DNA sequences spanning the ribosomal DNA repeat. By monitoring
the hybridization signals it was possible to determine the sequences
that were selectively represented in the fraction of nascent DNA
fragments, meaning that these sequences contained sites that function
as DNA replication origins in vivo (Fig. 1).
H]dC or [H]dT,
and the short nascent DNA fragments synthesized at origins of
replication located between the cross-links (zone 2) were
isolated by alkaline sucrose gradient centrifugation. The low molecular
weight fraction was purified by immunoprecipitation with anti-BrdUrd
antibody and used for hybridization with dot-blotted in excess DNA
probes. Hybridization signal was obtained only with probes located at,
or close to the origin of replication (zone 2), and not with probes
located far from the origin region (zones 1 and 3).
in the
single copy DHFR domain in Chinese hamster ovary cells(18) ,
and a number of other mammalian origins of
replication(19, 20, 21, 22, 23) .
The rationale behind the unrestricted nascent chain approach is
schematically represented in Fig. 2. Exponentially growing
cells, uniformly labeled with [C]dT, were
labeled with BrdUrd and [H]dC, or
[H]dT for 10 min. During this time any DNA chains
initiated at the beginning of the labeling period will grow to
approximately 20 kb; DNA chains initiated later will grow only to
fractions of this length and DNA chains initiated at the end of the
labeling period will be very short. Thus for each active replication
origin a set of nascent DNA fragments of different length, centered at
the origin site, will be synthesized. By hybridizing the different size
classes of nascent DNA chains to genomic probes, it is possible to
determine the area where DNA synthesis has initiated. Genomic DNA was
isolated and size-fractionated by centrifugation in alkaline sucrose
density gradient. The different size fractions were purified by a
second round of alkaline sucrose density gradient centrifugation and
immunoprecipitation with anti-BrdUrd antibody. Their specific
radioactivity, determined by their H/C ratio
was practically identical with that of control genomic DNA, uniformly
labeled under the same conditions. This was an indication that the
purification procedure had efficiently eliminated contaminating DNA.
The purified nascent DNA fragments were hybridized with dot-blotted
cloned unique DNA probes spanning the ribosomal DNA repeat. In this
experiment the probes adjacent to the origin would hybridize both with
the short and long nascent DNA fragments, while probes distal to the
origin would hybridize with the long DNA fragments only.
H]dC or [H]dT the nascent
DNA chains were size-fractionated by alkaline sucrose density gradient
centrifugation, purified by immunoprecipitation with anti-BrdUrd
antibody, and hybridized with dot-blotted in excess DNA probes. The
probes located close to a replication origin (probe 2)
hybridized with all size fractions, while probes located farther from
an origin (probes 1 and 3), hybridized only with the
longer, and not with the short nascent DNA
fragments.
H and not P and the specific
radioactivity of the fragments was lower than it would have been if we
had labeled them in vitro. However, in this way we were sure
that we had eliminated the effect of any contaminating genomic DNA and
detected the newly synthesized DNA only.
Mapping Replication Origins in the Rat rRNA Genes
Fig. 3shows the physical map of the rat ribosomal DNA
repeat and the positions of the DNA sequences we have used as probes.
In the first experiment the in vivoH-labeled
immunoprecipitated origin fraction, isolated after cross-linking of
exponentially growing rat cells with Trioxsalen, was hybridized to
dot-blotted fragments RrII and RrIV, spanning most of the rat ribosomal
DNA repeat. Both clones gave positive signals. Fragment RrII, which
contained the gene for 18 S rRNA and part of the adjacent
nontranscribed spacer upstream of the promoter region, gave a stronger
signal than RrIV, which contained the gene for 28 S RNA and part of the
nontranscribed spacer downstream of the 3`-end of the gene.
Furthermore, DNA probes subcloned from RrII and RrIV were dot-blotted
on membranes and hybridized with the origin DNA fraction. In this case
only probes Rr56, a 5.2-kb DNA sequence located upstream of the
promoter sequence, and Rr151, a 3.9-kb sequence located at the 3`-end
of the transcription unit, gave good hybridization signals (Fig. 4A). This showed that there may be two initiation
zones in the rat ribosomal DNA repeat. One of them is more pronounced
(which would probably mean that it is used more often) and is located
upstream of the promoter region, and the second one is less well
pronounced and spans the 3`-end of the 28 S RNA gene and part of the
adjacent nontranscribed spacer region. In an attempt to map more
precisely the position of the stronger initiation site upstream of the
5`-end of the gene, we cleaved pRr56 with the restriction endonucleases EcoRI, EcoRV, and SauI, isolated the
restriction fragments designated A, B, and C, and hybridized them with
the origin fraction. The strongest signal was obtained with fragment A.
Fragment C gave a very weak signal and fragment B gave a signal with
intermediate strength (Fig. 4B). These experiments were
performed with in vivo labeled [H]dT or
[H]dC nascent DNA fragments, and in both cases we
obtained absolutely the same result with the only exception that in the
case of the [H]dT-labeled origin fraction the
signals were between two and three times weaker, due to the lower
specific radioactivity of the [H]dT-labeled DNA.
In all experiments a 1.2-kb mouse DNA fragment containing the DHFR gene
sequence was blotted along with the ribosomal probes and simultaneously
hybridized to estimate the background level hybridization. These
experiments indicated that an origin of DNA replication was located
about 4 kb upstream of the promoter region of the rat rRNA gene, most
probably within the 838-base pair EcoRI/EcoRV
fragment A.
10
counts of nascent DNA. Filters were cut and counted. Results are
means of three experiments after subtracting the background counts. Bars represent standard deviation.
Mapping Replication Origins in the Human rRNA
Genes
Exponentially growing HeLa cells were cross-linked with
Trioxsalen and labeled as above. The nascent DNA fraction was isolated
and hybridized to five dot-blotted probes that span the length of the
human ribosomal DNA repeat. In this case we were able to detect the
presence of a replication origin in the nontranscribed spacer 5` of the
rRNA gene, but the results were not statistically significant because
of the weak hybridization signal. For an unknown reason HeLa cells
incorporated labeled precursors less readily than Guerin cells and the
specific radioactivity of the labeled DNA was low. Moreover, both the
cloned probes and the nascent DNA fragments were relatively short,
which could also lead to the low hybridization signal. To get more
conclusive results about the presence and location of replication
origins in the human ribosomal DNA repeat we applied the unrestricted
nascent chains growth technique in which longer nascent DNA fragments
are used. We consider this technique comparable to the restricted
nascent chains growth approach since in two different laboratories the
positions determined for the DHFR ori- by the two
techniques coincided(7, 18) . DNA from exponentially
growing HeLa cells, pulse-labeled with [H]dC and
BrdUrd, was size fractionated by two successive alkaline sucrose
density gradient centrifugations to obtain five DNA fractions,
relatively homogeneous in respect to chain length. 28 S and 18 S rRNA
were run as size markers, and it was calculated that the five fractions
had average lengths of 30, 15, 8, 4, and 1.5 kb. The nascent DNA
fractions were immunoprecipitated with anti-BrdUrd antibody. Five
cloned DNA fragments spanning the ribosomal DNA repeat, designated by
numbers 1 through 5 (Fig. 5), were dot-blotted in excess and
were hybridized with the five size fractions of the in vivo labeled nascent DNA to determine the relative abundance of the
cloned fragments in the fractionated nascent DNA. To compare the
results obtained with the different size fractions we had to normalize
them. This could be done either by using a constant amount of labeled
DNA, regardless of the size of the fragments, or by using increasing
amounts of DNA with increasing size of the fragments. In the first case
we would measure the hybridization capacity in nucleotides per dot,
while in the second case it would be expressed in chains per dot. We
tried both approaches and found that the first approach gave more
reproducible results. Therefore a constant amount of counts of the five
size fractions was used for hybridization. A 211-base pair long DNA
fragment from the 5`-end of the human -actin gene was dot-blotted
as a negative control and was hybridized with all size fractions. It
gave background hybridization with all of them. Probe 1, located at the
initiation site for transcription of ribosomal DNA, exhibited high and
uniform hybridization with all size fractions of nascent DNA. Probe 2,
located at the 3`-end of 28 S RNA, also gave a strong and reproducible
hybridization signal with all size fractions, although the absolute
strength of the signal was lower than with probe 1. The signal obtained
with probe 3, located in the middle of the nontranscribed spacer, was
very weak when hybridized with short nascent DNA fragments and
approached the strength of the signals obtained with probes 1 and 2
when hybridized with the longest nascent DNA fragments. This was an
indication that this probe was not located near an origin of
replication. Probe 4 gave a hybridization pattern similar to that of
probe 3, which showed that this sequence was also not located near an
origin of replication. Probe 5 showed a hybridization pattern similar
to that of probe 1 (Table 1). It is difficult to express the
relationship between fragment lengths, hybridization signals, and probe
locations in precise analytical form. Nevertheless, by empirically
arranging the nascent DNA fragments on the physical map of the
ribosomal DNA repeat, it was possible to obtain an alignment satisfying
the data in Table 1(Fig. 5). Thus, it could be argued
that since both probes 1 and 5, which are located 7 kb apart, hybridize
with size fragments 4-kb long, an initiation zone at least 4 kb in
length should be located between these two probes. On the other hand,
since probe 4, which is located 5 kb upstream of probe 5, does not
hybridize with the 8-kb nascent fragments, the length of this
initiation zone should not exceed 4 kb. This is an important finding
and the fact that the same size fragments hybridized with some probes
and did not hybridize with others showed that the initiation zones are
well defined and that no initiation occurs outside these zones. Similar
arguments lead to the conclusion that a second replication origin is
contained within an approximately 3 kb long sequence centered at the
3`-end of the transcription unit (Fig. 5).
H]dT. We obtained practically the same results
with the only difference that in the case of
[H]dT the specific radioactivity of DNA was
lower, because dT competed with BrdUrd.
G) of the initiation
zones. The analysis of the rat sequence reveals three local minima of
helical stability: around nt positions 180, 1180, and 1610,
respectively. The first and lowest minimum is located in the 838-base
pair EcoRI/EcoRV fragment that showed the strongest
hybridization signal with the origin fraction, and is therefore the
most likely origin-containing candidate. The human sequence displays
two local minima of helical stability, from nt -4822 to
-4678 and around nt position -4318. Both are situated
within the 1.38kb BamHI/SmaI fragment which in the in vitro replication reaction was evaluated as the most likely
origin-containing fragment(5) .
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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