(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) ()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.
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 [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).
The
unrestricted nascent chain growth technique was developed by Vassilev
and Johnson (8) and was used to map ori- 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.
Figure 2:
Diagram of the unrestricted nascent chains
growth experimental approach. Following pulse labeling with BrdUrd and
[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.
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 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.
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 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.
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,
[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.
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 (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) .
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.