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[Cancer Research 66, 5094-5103, May 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Differentially Active Origins of DNA Replication in Tumor versus Normal Cells

Domenic Di Paola1,2, Gerald B. Price1,{dagger} and Maria Zannis-Hadjopoulos1,2

1 McGill Cancer Center and 2 Department of Biochemistry, McGill University, Montreal, Quebec, Canada

Requests for reprints: Maria Zannis-Hadjopoulos, McGill Cancer Center, 3655 Promenade Sir William Osler, Room 710, Montreal, Quebec, Canada H3G 1Y6. Phone: 514-398-3536; Fax: 514-398-6769; E-mail: maria.zannis{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, a degenerate 36 bp human consensus sequence was identified as a determinant of autonomous replication in eukaryotic cells. Random mutagenesis analyses further identified an internal 20 bp of the 36 bp consensus sequence as sufficient for acting as a core origin element. Here, we have located six versions of the 20 bp consensus sequence (20mer) on human chromosome 19q13 over a region spanning ~211 kb and tested them for ectopic and in situ replication activity by transient episomal replication assays and nascent DNA strand abundance analyses, respectively. The six versions of the 20mer alone were capable of supporting autonomous replication of their respective plasmids, unlike random genomic sequence of the same length. Furthermore, comparative analyses of the endogenous replication activity of these 20mers at their respective chromosomal sites, in five tumor/transformed and two normal cell lines, done by in situ chromosomal DNA replication assays, involving preparation of nascent DNA by the {lambda} exonuclease method and quantification by real-time PCR, showed that these sites coincided with chromosomal origins of DNA replication in all cell lines. Moreover, a 2- to 3-fold higher origin activity in the tumor/transformed cells by comparison to the normal cells was observed, suggesting a higher activation of these origins in tumor/transformed cell lines. (Cancer Res 2006; 66(10): 5094-103)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chromosomal DNA is replicated in units termed replicons, whose average size varies from 10 to 300 kb, depending on the stage of development, growth conditions, or cell transformation status (1, 2). Each replicon contains one centrally placed functional origin (ori), where replication is initiated, with an estimated number of 104 to 106 replication origins per mammalian cell (1, 2). Control over the timing and frequency of initiation of DNA replication is exerted at the origin site (3, 4). We recently identified and tested a putative 36 bp mammalian origin consensus sequence capable of supporting autonomous replication of a plasmid after transfection into eukaryotic cells (5). Initiation of plasmid replication in vitro occurred within the consensus, whereas homologues of the consensus were found consistently at mammalian chromosomal sites of initiation of DNA replication as well as within CpG islands. Versions of the consensus functioned as origins of DNA replication in normal and malignant human cells; immortalized monkey and mouse cells; and normal cow, chicken, and fruit fly cells. Mutation analysis of the 36 bp consensus sequence suggested that an internal 20 bp sequence (20mer) is sufficient to act as a core origin consensus element. The distribution of the 20mer over 1 Mb human chromosomal DNA varied between approximately every 20 to 50 kb and was quantitatively and qualitatively (i.e., relative proximity to each other) similar to the distribution of the autonomously replicating sequence consensus sequence on Saccharomyces cerevisiae chromosomes (5).

Several variables regulate the activation of mammalian replication origins. These include the concentration and conformation of initiator proteins (6, 7), as well as the specific DNA sequences acting as replication origins having differential affinities for the six subunits of the origin recognition complex (6–8). Transcription factors may also play important roles in the initiation and timing of replication for different cell types (9, 10). Gene transcription, by way of transcriptional activation of certain gene loci in cells, has been linked to the initiation of DNA replication, which occurs earlier in S phase than in cells in which the same loci are not transcribed (11, 12). Chromatin structure and nuclear organization are also essential for the spatial positioning and interaction of replication origins with initiator proteins, thus regulating origin activation (13–15). Furthermore, epigenetic components leading to protein or DNA modification, such as DNA methylation (16, 17), histone modification (18), and nucleotide pool levels (19) have been postulated as determinants of replication origins. Deregulation of any one of these variables brought about by cellular transformation would be expected to result in altered DNA replication as is the case during embryonic development (20, 21). Thus, cellular transformation may modify the regulation of origin activation, resulting in differential origin usage between normal and transformed cells.

Cellular transformation and tumor progression are thought to be a return to the early stages of embryonic development (3). DNA fiber autoradiography measurements indicated that the average replicon size was decreased in transformed cells by comparison to normal cells (22, 23), suggesting the existence of tumor-specific and malignancy-regulated origins. Furthermore, a 2- to 10-fold increase of single-strand nuclease sensitive regions was found in transformed cells, consistent with more origins being activated (24). In addition, some mammalian replication origins, including the c-myc origin, were approximately twice as active in HeLa cells as in human normal skin fibroblasts (NSF; ref. 25). Although the relative nascent DNA abundance across 12.5 kb of the human c-myc locus had a similar distribution, a 2-fold higher abundance was found in HeLa than in NSF cells at the majority of the initiation sites tested across the c-myc locus (26). A comparison of origin activities between the isogenic pair of normal human embryo lung fibroblasts (WI38) and their SV40-transformed immortal variant [WI38(SV40)] indicated a similar distribution of nascent DNA with 2-fold higher abundance in the transformed [WI38(SV40)] than in the normal (WI38) cells at the majority of sites tested across the c-myc locus, eliminating the possibility that cell type effects were responsible for the observed differences in origin activities in HeLa and NSF cells (27). Overall, these findings suggested that cellular transformation might induce greater frequency of initiation of origins in certain loci.

A polarity or position change of initiation of DNA replication was also observed with malignant transformation (28). Fundamental differences in the organization of DNA replication sites in normal versus immortalized cell lines have been reported (29), but other studies found no such differences (30, 31). Different chromatin organization and rearrangements as well as different nuclease sensitivity between normal and transformed cells taking place during the progression of S phase cells have also been reported (32, 33). Moreover, the replication timing of homologous loci was found to be more asynchronous in samples derived from transformed cells relative to their normal counterparts (34).

In this study, we searched for tumor-specific or tumor-activated origins, using the 20mer as bait to identify replication origins, and comparatively analyzed the nascent DNA abundance across these sites in two normal and five tumor cell lines. We located six versions of the 20mer in a region spanning ~211 kb on human chromosome 19q13 and tested their ability to act as replication origins by ectopic and in situ replication assays. The results showed that these six versions of the 20mer consensus were able to support the ectopic autonomous replication of their respective plasmids and also served as chromosomal replication origins in situ. Furthermore, tumor/transformed cells had an ~2- to 3-fold higher origin activity at the 20mer sites than normal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sequence analyses. A ~211 kb region of chromosome 19q13 (Genbank accession no. NT_011109) was scanned for the presence of a 20 bp consensus sequence (5), using the program fuzznuc of the EMBOSS suite of software, allowing two mismatches and no gaps (Table 1 ).3


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  		Table 1. Location of the 20 bp human consensus sequence in a ~211 kb region of human chromosome 19q13

 
Cloning and preparation of constructs. Plasmid constructs used in the transient replication assays were prepared by annealing complementary oligonucleotides (Table 2 ) into the BamHI and EcoRI sites of the pBluescript vector (Stratagene, La Jolla, CA) using a standard protocol (35). Supercoiled plasmid DNA of the resulting clones as well as the pM1 SEAP vector (Roche Molecular Biochemicals, Indianapolis, IN) was prepared using the Qiagen Maxiprep kit (Qiagen, Mississauga, ON, Canada) according to specifications of the manufacturer, and sequenced.


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  		Table 2. Complementary oligonucleotides used to prepare constructs to test versions of the 20 bp human consensus sequence for autonomous replication in the in vivo DNA replication assay

 
Cell culture. HeLa, NSF (primary NSFs), PC-3, HCT 116, LS174T, WI38 (human lung embryo fibroblasts), and WI38 VA13 2RA (WI38 transformed with SV40 virus) were acquired from American Type Culture Collection (Manassas, VA) and cultured in {alpha}-MEM supplemented with penicillin (100 µg/mL), streptomycin (100 µg/mL), 1 mmol/L L-glutamine, tylosin (8 µg/mL), and 10% (v/v) fetal bovine serum. When the cells reached 30% to 50% confluence, they were harvested for the isolation of nascent DNA, whereas upon reaching 100% confluence, they were serum-starved for 48 to 72 hours and harvested for the isolation of genomic DNA.

Episomal DNA replication (DpnI resistance) assay. For transfections, HeLa cells were seeded in six-well plates at a density of 3 x 104 per well, and ~16 hours later were transfected with 3 µg supercoiled plasmid DNA (2 µg of each construct in Table 2 and 1 µg pM1 SEAP), using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) as per instructions of the manufacturer. At 72 hours post-transfection, low molecular weight DNA was isolated and digested with DpnI, the DpnI-digested and undigested DNA were used to transform the DH5{alpha} strain of Escherichia coli and the relative in vivo DNA replication of each transfected plasmid was determined by counting the number of colonies in a bacterial retransformation assay, as previously described (36–38). The levels of secreted human placental alkaline phosphatase, determined by the SEAP Reporter Gene Assay kit (Roche Molecular Biochemicals), as per specifications of the manufacturer, were used to normalize the transfection efficiency.

Isolation of genomic DNA. Genomic DNA was isolated using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma, Oakville, ON, Canada), as per instructions of the manufacturer.

Isolation of nascent DNA. Nascent DNA was prepared using the {lambda} exonuclease method, as previously described (26), with the following modifications: The {lambda} exonuclease–digested samples were heated at 100°C for 3 minutes, then immediately subjected to electrophoreses on a 2% agarose gel. DNA was visualized by staining with 0.02% (w/v) methylene blue (Sigma) and the origin-containing nascent DNA, ranging between 350 and 1,000 bp in size was excised from the gel, purified with the Sephaglas BandPrep kit (GE Healthcare, Piscataway, NJ), as per instructions of the manufacturer, and resuspended in TE.

Real-time PCR quantification analyses. PCRs were carried out in a total volume of 20 µL with 5 µL genomic or nascent DNA, using the LightCycler (Roche Diagnostics), as previously described (39, 40). The sequences and amplification conditions for all primer sets are shown in Table 3 . Genomic DNA (1, 2, 3, and 4 ng) from NSF cells was used to generate the standard curves needed for quantification of the PCR products. A negative control without template DNA was included with each set of reactions. PCR products were resolved on 2% agarose gels, visualized with ethidium bromide, and photographed with an Eagle Eye apparatus (Speed Light/BT Sciencetech-LT1000). No extraneous bands were generated with any of the primer sets.


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  		Table 3. Sequences and amplification conditions of primers used for real-time quantitative PCR on the LightCycler

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Occurrence of 20mer homologues in a ~211 kb region on human chromosome 19q13 and in the lamin B2 locus. A chromosomal region of ~211 kb located on human chromosome 19q13 was scanned for the presence of the 20mer consensus (5), using the program fuzznuc of the EMBOSS suite of software. Setting the variables at two mismatches and no gaps, six 20mers were detected at a level of 90% homology (Table 1). The well-characterized lamin B2 origin region was also used as a control. We located two different versions of the 20mer included in the origin of bidirectional replication (OBR) of the lamin B2 locus, one copy on the plus strand (M94363 nucleotides 3910-3929) and the other on the minus strand (M94363 nucleotides 3953-3934), both at a level of 75% homology. A diagram of the human lamin B2 locus and of the ~211 kb region of chromosome 19q13 is shown (Fig. 1A and B ).


Figure 1
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  		Figure 1. A, loci map of the lamin B2 locus (horizontal black line) showing the lamin B2 and PpvI genes, from left to right, respectively (gray arrows pointing to the direction of transcription). The position of primer sets LB2 and LB2C1 used in real-time PCR is indicated (black arrows pointing upwards). The location of the lamin B2 origin (L, light gray circle) and of a negative control region located ~4 kb upstream of the origin are shown (C, dark gray circle). From the first to last primer set, the region shown is represented by nucleotides 1 to 4,070 (accession no. M94363). B, loci map of an ~211 kb region on human chromosome 19q13 (horizontal black line), showing the location of the six 20mers with matches to the 20 bp consensus sequences (90% homology; refer to Table 1). Gray arrows, genes pointing to the direction in which they are transcribed (GPR32P, GPR32, ACPT, MGC13170, MGC45922, KLK1, KLK15, KLK3, KLK2, KLK4, LOC390956, KLK5, and KLK6 genes from left to right). Black arrows pointing upwards, primer sets used for real-time PCR. Light gray circles (1-6), location of their respective 20mers (containing a version of the 20 bp consensus sequence); dark gray circles (C, C1, C2, C34, C56), negative control regions (at least 6 kb away from any of the six 20mers). From the first to last primer set, the region shown is represented by nucleotides 23,516,094-23,727,937 (accession no. NT_011109). C, episomal replication activity of the various versions of the 20 bp consensus sequences (20mers 1-6). Histogram plot of the number of bacterial colonies produced after transformation of E. coli with DpnI-digested Hirt extracts of HeLa cells transfected for 72 hours with one of the following plasmids (refer to Table 2): pA3/4, p20mer1-6, p20merC, pLB2, pLB2C1, and pBluescript. The average number of DpnI-resistant colonies per plate was corrected for the amount of DNA recovered and normalized to the number of colonies obtained with pA3/4 (positive control), which was taken as 100%. The number at the bottom of each column denotes the average number of colonies obtained from three experiments. Columns, average of three experiments done in triplicate; bars, SD.

 
Autonomous replication activity of plasmids containing the 20mer. The ability of the 20mer versions, found on human chromosome 19q13 and in the lamin B2 origin region, to confer autonomous replication activity when cloned into a plasmid was analyzed by the DpnI resistance assay, which is an indicator of semiconservative DNA replication, as previously described (36–38).

HeLa cells were transfected with one of the following constructs (Table 2): p20mer1-6, containing versions of the 20 bp consensus sequence found in the ~211 kb region of human chromosome 19q13; p20merC, containing genomic sequence found within the ~211 kb region of human chromosome 19q13 that does not contain a version of the 20 bp consensus (negative control); pA3/4, containing a version of the 36 bp consensus (positive control; ref. 5); pLB2, containing a version of the 20 bp consensus included in the OBR of the lamin B2 locus; pLB2C1, containing genomic sequence found within the lamin B2 locus that does not contain a version of the 20 bp consensus (negative control for the lamin B2 locus; ref. 41); pBluescript vector without an insert (negative control); pM1 SEAP vector was co-transfected with each construct to normalize for the transfection efficiency; finally, a mock transfection without a construct was done as an additional negative control. At 72 hours post-transfection, plasmid DNA was isolated, digested with DpnI, and the DpnI-digested DNA was used to transform E. coli. After 18 hours, the number of colonies produced was counted, corrected for the amount of DNA recovered, and related to the positive control reaction of construct pA3/4, which was taken as 100% (Fig. 1C). As expected of mammalian replication origins, which are activated only once per cell cycle, a relatively small number of colonies (ranging from 14 to 33) was produced, on average, by even the most efficiently replicating plasmid (pA3/4, 33 colonies). Furthermore, the "low" signal would be the result of weaker replication activity by the basal consensus replication element (20mer) alone than would be expected if the insert contained additional sequences that would increase replication efficiency. The plasmids containing the 20mers 1, 2, 4, 5, and 6 replicated almost as efficiently as pA3/4, which displayed the most efficient replication, as before (5). On the other hand, p20mer3 replicated at 40% efficiency by comparison to pA3/4, suggesting that the 20mer3 version of the consensus was a less efficient replication origin than 20mers 1, 2, 4, 5, or 6. In contrast, 20merC was unable to confer autonomous replication to its plasmid, suggesting that 20mers 1 to 6, representing versions of the origin consensus sequence, could act as replication initiation sites. This notion was corroborated by the result obtained with pLB2, containing the peak origin region of the lamin B2 locus, which was able to replicate autonomously at nearly the same efficiency as p20mers 1, 2, 4, 5, and 6, unlike pLB2C1, which was unable to confer autonomous replication to its plasmid. Similarly, no bacterial colonies were obtained with DpnI-digested DNA recovered when either the backbone vector (pBluescript) had been transfected, as before (38), or from the mock transfections.

Copy number of 20mer sites. To assess the copy number per haploid genome at all regions examined in this study, equal amounts of genomic DNA from each cell line was amplified by real-time PCR. Figure 2A, top , shows a representative ethidium bromide–stained 2% agarose gel of the expected amplification product of 194 bp obtained with the M20mer2 primer set. The results (Fig. 2A, bottom) were normalized by making NSF equal to one copy per haploid genome and show that in all cell lines M20mer2 was present at approximately one copy per haploid genome. Similar results were obtained for all primer sets (data not shown), indicating that the primer sets amplifying all the regions examined in this study were present at one copy per haploid genome in each cell line.


Figure 2
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  		Figure 2. A, copy number per haploid genome of 20mer2 within the ~211 kb region on human chromosome 19q13 in several normal and transformed cell lines. Top, PCR amplification product (194 bp) produced by the M20mer2 primer set. Template DNA was as follows: lanes 1 to 3, genomic DNA from NSF [1, 2, and 3 ng, respectively; necessary to build the standard curve used for quantification of DNA at M20mer2 by real-time PCR; positive control (+ve)]; lanes 4 to 10, 10 ng genomic DNA from HeLa (H), NSF (N), PC-3 (P), HCT 116 (HC), LS174T (L), WI38 (W), WI38(SV40) (WS); lane 11, water [no template DNA added, which is necessary to check for contamination; negative control (–ve)]. All bands were 194 bp in size. Bottom, histogram plot of copy number/haploid genome of the cell lines used in the study. The results were normalized by making NSF equal to one copy/haploid genome. Columns, average of at least two experiments done in triplicate; bars, SD. (Note that this is a representative graph; the copy number of all the primer sets used in this study was measured; see Table 3.) B, nascent DNA preparation. Lane 1, 1 kb Plus DNA Ladder; lane 2, ~600 ng total cellular (DNA + RNA) sheared with a fine needle (26G3/8); lane 3, ~1,200 ng sheared and denatured total cellular (DNA + RNA); the single band was the internal control, linear pCR-XL-TOPO (~50 ng); lane 4, sheared total cellular (DNA + RNA) and internal linear control digested by {lambda} exonuclease; lane 5, the nascent DNA sample after further treatment with RNase A; lane 6, HindIII-digested {lambda} phage DNA marker. The nucleic acids were separated on 1% agarose native gel. C, assessment of quality of nascent DNA, exemplified by the lamin B2 origin, in the cell lines shown [HeLa, NSF, PC-3, HCT 116, LS174T, WI38, and WI38(SV40)]. Histogram plot of the nascent DNA abundance (ng), measured by real-time PCR, at the lamin B2 peak (dark gray) and a non–origin-containing (control) region, located ~4 kb downstream (light gray). Columns, average of at least two experiments done in triplicate; bars, SD.

 
Nascent DNA abundance. The above results allowed quantitative comparisons between the nascent DNA abundance among the various cell lines, thus permitting measurements of replication origin activity.

The {lambda} exonuclease digests the 5' phosphorylated parental and broken DNA strands, but the nascent DNA strands bearing 5' RNA primers are resistant to digestion by this enzyme (42). Figure 2B is representative of all nascent DNA preparations done for each cell line. Total cellular DNA (together with total RNA) was isolated and sheared with a fine needle. The average size of the sheared total cellular DNA was ~30 kb, seen as a band on a 1% agarose gel, whereas the smear of lower molecular weight represented RNA (Fig. 2B, lane 2). Phosphorylation and {lambda}-exonuclease digestion were monitored by an internal control of dephosphorylated linear plasmid (Fig. 2B, lane 3; refs. 26, 27); the plasmid DNA was completely phosphorylated and digested by {lambda} exonuclease (Fig. 2B, lane 3 versus lane 4), and residual RNA (Fig. 2B, lane 4) was removed by RNase A digestion (Fig. 2B, lane 5).

To eliminate any Okazaki fragments that might contaminate the nascent DNA preparations, the samples were subjected to electrophoresis on agarose gel, stained with methylene blue, and only nascent DNA ranging between 350 to 1,000 bp was excised and purified.

To assess the quality of nascent DNA samples, the distribution of nascent DNA from an origin region associated with the lamin B2 locus was quantified. This origin was shown to be active in a number of different proliferating human cells (26, 27, 41, 43, 44). Nascent DNA was measured at two reference points, one located at the center of the origin (LB2 primer set = peak activity), the other located ~4 kb away (LB2C1 primer set = background activity), using the primer sets described by Ladenburger et al. (45). The ratio of highest to lowest abundance in the region to be studied is indicative of the quality of the nascent DNA preparation and signal-to-noise estimates (26, 27, 44). Moreover, a ratio of signals from the initiation site to distant sites in noninitiation regions of ≥10 is indicative of good quality nascent DNA (46). The ratio of highest abundance at the lamin B2 peak region to lowest abundance at the lamin B2 control region ranged from 10.5 to 21.0, indicating good quality nascent DNA preparations for all cell lines (Fig. 2C), and thus suitable for mapping replication origins in vivo. These samples were used to measure the nascent DNA abundance across the ~211 kb region of human chromosome 19q13, in each of the tumor/transformed and normal cell lines. Similar results were obtained when these experiments were done using sheared and sonicated genomic DNA, yielding a distribution of fragment lengths similar to that in the population of origin-proximal nascent DNA, verifying that the efficiency of the different primer sets was the same as when tested with template DNA of high molecular weight (used to construct the standard curves; data not shown).

Origin activities at 20mer sites across a ~211 kb region of human chromosome 19q13 in tumor/transformed and normal cell lines. Origin activity in vivo was measured in five tumor/transformed and two normal cell lines, by quantification of the nascent DNA abundance across the ~211 kb region on human chromosome 19q13 (Fig. 3 ). Each of the 23 primer sets used amplified a fragment of ~150 to 300 bp in size. Each of the six 20mers (Fig. 3, top, light gray circles labeled 1-6) that represent putative replication initiation sites were amplified using three primer sets: M amplifies a region containing a version of the 20 bp consensus sequence; L amplifies a region located 5' (upstream) of the M region; and R amplifies a region located 3' (downstream) of the M region. In addition, five negative control regions (Fig. 3, top, dark gray circles labeled C, C1, C2, C34, C56) were used, representing regions not containing replication origins, thus allowing us to measure the lowest abundance of nascent DNA in the ~211 kb region being examined. C1 designates a region located ~6 kb from 20mer1, C2 one located ~6 kb from 20mer2, C34 one located at least 6 kb from 20mer3 and 20mer4, C56 one located at least 6kb from 20mer5 and 20mer6, and C designates a region located at least 44 kb away from any 20mer. Within the lamin B2 locus, besides the positive region (LB2) containing two versions of the 20 bp consensus sequence at 75% homology, the negative region (LB2C1) also contains one at 75% homology, but it is not at an origin site, suggesting that the presence of a 20mer is not sufficient for origin activity. To further assess whether all 20mers with homology to the 20 bp consensus correlated with origin activity in our region of interest, two of the negative control regions (20merC2 and 20merC34) were designed to contain versions of the 20 bp consensus with 75% homology, compared with 20mers 1 to 6, containing versions of the 20 bp consensus with 90% homology; the remaining negative control regions (20merC, 20merC1, and 20merC56) did not contain any version of the 20 bp consensus.


Figure 3
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  		Figure 3. Nascent DNA abundance at the ~211 kb region of human chromosome 19q13 containing the 20mers. Histogram plots of the quantification by real-time PCR of nascent DNA abundance (ng) across the ~211 kb region of human chromosome 19q13 (horizontal black line) of five tumor/transformed cell lines (gray columns) and two normal cell lines (white columns). The location of the primers (see Table 3) used for the amplification of 20mers 1 to 6 (light gray circles numbered 1-6) and the control regions (dark gray circles labeled C, C1, C2, C34, C56) are indicated (black arrows pointing upwards). The distances between the 20mer primer sets are as follows: 20mer6 to 20mer5, 4.6 kb; 20mer5 to 20mer1, 30.8 kb; 20mer1 to 20mer2, 87.9 kb; 20mer2 to 20mer4, 83.5 kb; 20mer4 to 20mer3, 3.8 kb; and those of the control regions are: 20merC1 to 20mer1, ~6 kb; 20merC2 to 20mer2 ~6 kb; 20merC34 to 20mer3 and 20mer4, at least 6 kb; 20merC56 to 20mer5 and 20mer6, at least 6 kb; finally, 20merC, at least 44 kb from any 20mer. Each of the 23 primer sets amplifies a region of ~150 to 300 bp in size: M, a region containing the 20mer; L, a region located ~300 to 400 bp 5' (upstream) of the M region; R, a region located ~300 to 400 bp 3' (downstream) of the M region. Columns, average of at least two experiments done in triplicate; bars, SD.

 
The nascent strand abundance across the ~211 kb region of human chromosome 19q13 was determined in the same preparation of short nascent DNA and normalized to that of an internal reference, the lamin B2 locus, to control for the possibility of a greater recovery of nascent DNA from the transformed cells compared to the normal ones. Specifically, amplification of nascent DNA with primer set LB2C1 (background activity compared with lamin B2 peak region, primer set LB2) gave baseline values that were used to normalize results from all the nascent DNA preparations of all cell lines, permitting comparison of data between different preparations and different cell lines.

Histogram plots of the nascent DNA abundance measured at the various regions in the cell lines used (Fig. 3, bottom) show a peak of origin activity at each of the six 20mers (20mers 1-6), diminishing (75-25% less origin activity) at regions immediately flanking the 20mers and reaching background levels (≤10% origin activity) at the five negative regions (C, C1, C2, C34, and C56) located at least 6 kb away from any of the six 20mers. These data indicate that 20mers 1 to 6 are located at sites of initiation (origins) of DNA replication. Taken together with the observation that two of the negative regions (20merC2 and 20merC34) with 75% homology to the consensus sequence contained background amounts of nascent DNA, the data suggest that the 20 bp consensus sequence designates the location of potential chromosomal replication origins.

The results also indicated a 2- to 3-fold higher origin activity in the tumor/transformed cell lines compared with the normal ones, suggesting a tumor-related activation of these origins located on human chromosome 19q13. Use of the isogenic pair of WI38 and WI38(SV40) showed that the origins were at least twice as active in the transformed compared to the normal cell line, ruling out the possibility that the observed increased frequency of initiation in tumor/transformed cell lines might be due to cell type. In all the cell lines examined, the highest abundance of nascent DNA was located at the position of primer set M20mer1, suggesting that 20mer1 coincided with the major initiation start site within the ~211 kb region located on human chromosome 19q13. Also, the lowest abundance was located at the position of primer set 20merC, which is most distant from any 20mer, indicating that no origin was located at that region (Fig. 3). Throughout the entire region, the ratio of highest abundance at primer set M20mer1 to lowest abundance at 20merC for all cell lines ranged from 21.2 to 36.6, confirming the good quality of the nascent DNA preparations. Furthermore, all six 20mers exhibited ratios of highest (peak activity) to lowest (background activity) abundance of >10, confirming that these are true start sites of DNA replication (46).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Homologues of the mammalian 20 bp consensus sequence on human chromosome 19q13 support autonomous replication of plasmids and coincide with chromosomal replication origins. In this study, we located six versions of the 20mer (at a level of 90% homology) on human chromosome 19q13 as well as a version of it included in the OBR of the lamin B2 locus (at a level of 75% homology) and tested them for both ectopic and endogenous (in situ) replication activity. These versions of the 20mer alone were sufficient for autonomous replication of their respective plasmids after transfection into HeLa cells, although not all versions replicated as efficiently as A3/4, a version of the original 36 bp consensus sequence (5) that was used as a positive control. In contrast, random 20 bp genomic sequences were unable to support autonomous replication. Using real-time PCR to quantify the abundance of nascent DNA, in five tumor/transformed and two normal cell lines, we found that the six 20mers on human chromosome 19q13 corresponded to chromosomal origins of DNA replication in situ, as does the 20mer present in the well-characterized lamin B2 origin, in all cell lines. A peak of origin activity at each of the six 20mers was observed, whereas less activity was found at regions immediately flanking them and background levels of origin activity were found at five negative regions located at least 6 kb away from any of the six 20mers. Furthermore, these origins were 2- to 3-fold more active in the tumor/transformed cells compared with the normal cells.

The replication origin associated with 20mer1 had a consistently higher signal-to-noise ratio than those associated with the other five 20mers in all cell lines examined, suggesting that the frequency of origin usage at the region of 20mer1 was higher than those at the other 20mer sites. Potentially, the origin at 20mer1 may be activated in more cells per population compared with the origins located at 20mers 2 to 6. Alternatively, there may be other potential initiation sites in the vicinity of the origins associated with 20mers 2 to 6, hence producing a broader and flatter peak by comparison to the origin associated with 20mer1. The latter might apply to the origins located at 20mers 3 and 4 and 20mers 5 and 6, due to their relatively close proximity to one another. As for the origin located at 20mer2, the possibility that additional initiation sites exist cannot be ruled out. Interestingly, these 20mer origins are not as active as the lamin B2 origin, which seems to be used in more cells per population than the six 20mer origins, possibly due to the former lying in a constitutively expressed gene domain, coding for a housekeeping protein (47).

Comparison of the ectopic and endogenous replication data (Figs. 1C and 3, respectively) revealed differences in the replication efficiencies of the six 20mers and suggested that the surrounding chromosomal sequence may be involved in regulating replication origin activity. Specifically, the episomal assay showed that 20mers 1, 2, 4, 5, and 6 conferred autonomous replication to their respective plasmids with approximately equal efficiency, whereas the plasmid bearing 20mer3 replicated with approximately half of that efficiency, suggesting that additional enhancing sequences may be required. The in situ chromosomal assay, on the other hand, showed that 20mer1 exhibited a 2-fold higher origin activity than 20mers 2 to 6, whose activities were all approximately equal. Overall, the data indicate that the presence of a 20mer designates a potential start site of DNA replication, but the determinant of whether it acts as one may be its accessibility for the assembly of a pre-replication complex.

The fact that although there are examples of the 20mer with 75% homology elsewhere in the ~211 kb region as well as in the lamin B2 negative region (LB2C1) that do not represent initiation sites suggests that the presence of the 20mer alone is not sufficient for determining replication origin activity in mammalian cells. It rather designates the location of potential origins and only when all the conditions are optimal and the configuration of the chromatin is appropriate does it lead to the licensing and activation of the origin.

Origin activities in normal versus tumor cells. Comparative analysis of the activity of chromosomal replication origins corresponding to the six versions of the 20 bp consensus sequence between five tumor/transformed cell lines and two normal cell lines confirmed that these 20mers were active initiation sites in all cell lines and also revealed a 2- to 3-fold higher origin activity in the tumor/transformed cells by comparison to the normal ones, across the ~211 kb region on human chromosome 19q13, corroborating the findings of previous studies (25–27). Furthermore, use of the isogenic pair of WI38 and WI38(SV40) confirmed these results, ruling out the possibility that the observed increase in origin activity was due to cell type effects. Use of these isogenic cell lines also indicated that the unregulated increase in replication initiation occurs at the transformation stage, before further advancement to cancer, causing replication stress, which may lead to DNA double-strand breaks and activation of the DNA damage checkpoint, increased genomic instability, and tumor progression (48, 49). Thus, increased origin firing may be an early event in the stepwise progression to cancer.

A possible explanation for the differential origin activities observed between the tumor/transformed and normal cells might be that some origins fire more than once. It has been speculated that the early events of genomic instability in a cancer cell might entail unregulated origin firing, providing substrates for genetic recombination and further amplification (50). Alternatively, the differential origin activities might be due to the six initiation sites not always being used in normal cells, but used in a higher percentage of tumor/transformed cells. It is conceivable that in normal cells, at least during some S phases, the DNA across this ~211 kb region on human chromosome 19q13 might be replicated by upstream or downstream origins flanking this region.

Previous studies have suggested that there are at least two types of changes in the activation of replication origins during cell transformation and malignancy: an increase in origin activity at some loci (25–27) and the activation of origins that are silent in normal cells (22, 23). The origins examined in this study fall under the category of those whose activity is increased in tumor/transformed cells by comparison to normal ones. Thus, it seems that there are at least three subsets of origins: those that are normal and remain unchanged, those with increased origin activity in transformed/immortalized or malignant cells, and those that are activated uniquely in tumor cells.

In summary, we have found six versions of the 20 bp consensus in a region of ~211 kb of chromosome 19q13, which confer autonomous replication of a plasmid and act as chromosomal origins of DNA replication. We also found a differential origin activity between normal and transformed cells at the chromosomal sites that contain these 20mer sequences. Thus, the 20mer seems to be an important variable in designating the location of replication origins. There may be other potential initiation regions marked by the presence of a 20mer with less homology to the consensus, as observed with the lamin B2 origin. Furthermore, the possibility of the existence of an entirely different subset of origins that are devoid of the consensus sequence cannot be ruled out.


   Acknowledgments
 
Grant support: National Cancer Institute of Canada-Canadian Prostate Cancer Research Initiative (M. Zannis-Hadjopoulos), Cancer Research Society (G.B. Price), and a studentship from the McGill University Cancer Consortium Training Grant in Cancer Research of the Canadian Institutes of Health Research (STP-53888, D. Di Paola).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: {dagger}This work is dedicated to the memory of Dr. Gerald B. Price, whose expert intellectual and technical advice and most of all friendship will be greatly missed. Back

3 http://bioweb.pasteur.fr/seqanal/interfaces/fuzznuc.html. Back

Received 11/ 2/05. Revised 3/ 8/06. Accepted 3/17/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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D. Di Paola, E. Rampakakis, M. K. Chan, D. N. Arvanitis, and M. Zannis-Hadjopoulos
Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation
Nucleic Acids Res., January 11, 2010; (2010) gkp1192v1.
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