From the
McGill Cancer Centre, McGill University, Montreal, Quebec H3G 1Y6, the ||Department of Internal Medicine, the Ottawa Hospital-General Campus, Ottawa, Ontario K1H 8L6, and the **Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
Received for publication, July 12, 2002 , and in revised form, March 12, 2003.
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ABSTRACT |
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INTRODUCTION |
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Our studies with small fragments of DNA which can support autonomous replication of a plasmid in mammalian cells (6, 7, 8, 9, 10) encouraged us to look further for putative replicator sequences. We report here the identification and testing of a putative consensus sequence that will aid in identification of initiation sites (origins) of DNA replication in mammalian and higher eukaryotic cells. We used four mammalian autonomously replicating sequences containing -satellite sequence and a reiterative process between pairs of African green monkey and human sequences to minimize derivation of an
-satellite consensus. The resultant consensus sequence was 36 bp.
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EXPERIMENTAL PROCEDURES |
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CpG Island ClonesTwo clones (CP9, HS14C3R Locus; and 6K, HS8F7F Locus) were obtained from the UK HGMP Resource Centre, Cambridge, UK. The sequence homolog in each clone is CATCGAAGCGCTTGAAATCTCCACTTACAAATTCC for CP9, and CCTCAAAGCGCTTGAAAATCTCCACTTGCAAATTCC for 6K. The CpG clones are in the vector pGEM-5Zf(-).
Plasmid DNAAll plasmid DNA clones were propagated in bacteria in LB medium containing 100 µg/ml ampicillin, and large scale amounts of supercoiled plasmid DNA, essential for autonomous replication assays in vivo and in vitro, were prepared using the Qiagen tip 500 columns according to the manufacturer's specifications (Qiagen).
Cell Culture and TransfectionHeLa cells were obtained from the American Type Culture Collection and cultured in Alpha-modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Flow Laboratories). The cultures were maintained in a 37 °C incubator containing an atmosphere of 10% CO2 + air. All normal primary cells (WI38 human embryo lung fibroblasts, bovine embryo kidney fibroblasts, and chicken embryo fibroblasts) were obtained from BioWhittaker and maintained in culture as described for HeLa cells. Drosophila S2 cells were maintained in Schneider's Drosophila medium (Invitrogen) with 10% heat-inactivated fetal calf serum supplemented with glutamine, asparagine, and penicillin/streptomycin. The cells were sealed in Nunc tissue culture flasks and incubated at room temperature in the dark.
Transfections were carried out as described previously (12, 13). Cells were cultured at 1 x 104 cells/cm2 in tissue culture flasks, T25 (Nunclon) overnight before transfection with 5 µg of supercoiled plasmid DNA, prepared using the calcium coprecipitation method (14). After transfection, the cells were grown for 24 h in medium containing bromodeoxyuridine (BrdUrd), as described previously (7, 12, 15). Plasmids were recovered by Hirt lysis (16), loaded onto CsCl gradients (initial refractive index 1.408), and centrifuged as described previously (7, 12). An aliquot of each fraction was either dotor slot-blotted onto a Gene-Screen Plus membrane (PerkinElmer Life Sciences), hybridized to 32P-labeled vector (pCRscript or pBluescript) DNA, exposed to an imaging plate, and quantified by densitometry performed using a PhosphorImager (Fuji BAS 2000).
In some cases, episomal DNA was recovered by Hirt lysis 3 days after HeLa cells were cotransfected with plasmids containing various versions of the consensus sequence and an expression plasmid carrying the luciferase gene, pRSVLUC (17). The low molecular weight DNA was digested with DpnI and then used to transform the DH5 strain of Escherichia coli in a bacterial retransformation assay, as described previously (7, 9, 10). Some of the transfected HeLa cells were used to determine variations in cell density and efficiency of transfection, by measuring levels of luciferase as described previously (17). The levels of luciferase were used to normalize the transformed bacterial colonies detected on LB agar plates containing 100 µg/ml ampicillin. In some cases, we also used pCMV/
-galactosidase (Applied Biosystems) and a
-galactosidase assay kit (Invitrogen).
In Vitro DNA ReplicationThe cell-free replication assay was adapted from the method described previously (11) and as performed previously (18). The earliest labeled fragment method was performed using the in vitro DNA replication system, as described previously (11, 18). In brief, the in vitro reactions were stopped at 4 and 8 min of incubation, the DNA products were digested with DdeI and PvuII and then separated on a 1.5% agarose gel in 1 x TAE buffer. The gel was dried and exposed to a PhosphorImaging plate. Incorporation of [-32P]dCTP and [
-32P]dTTP into each fragment was quantitated by densitometry of a PhosphorImager screen using the Fuji BAS 2000 analyzer and expressed as incorporation/kb of DNA.
Stability of pYACneo Constructs Containing Origins and the A3/4 Consensus SequenceAfter transfection of pYACneo (Clontech) constructs, including pYACneo with the A3/4 insert placed at the EcoRI site, clones of HeLa cells that were resistant to G418 were maintained in continuous culture. A fluctuation assay, as described previously (10), was performed upon six independent HeLa cell clones maintained for more than 40 cell doublings in medium containing 400 µg/ml G418.
Mutagenesis of A3/4 Version of the Consensus SequenceThe Gene-Morph PCR mutagenesis kit (Stratagene) was used according to the manufacturer's instructions to introduce random mutations in the 36-bp consensus sequence known as A3/4. One of the original clones containing A3/4 in pCRscript was used with T3 and M13 universal primers to prepare the product for ligation into pCRscript at the SmaI site. After transformation of DH5 competent cells (Invitrogen), numerous colonies were isolated and sequenced using the T7 primer in an ABI Prism 3700 DNA Analyzer (PerkinElmer Life Sciences). Among more than 100 clones examined, we found 38 variants with one or more mutations in the 36-bp region comprising A3/4 (see Table VI). After identification of these 38 variants, plasmid preparations were made using Qiagen HiSpeed Mini or Midi Kits. Then, an equimolar pool that contained DNA from the 38 variants plus A3/4 was used to transfect HeLa cells, as described above. After isolation of the low molecular weight DNA fraction by Hirt lysis, the DNA was digested with DpnI to remove unreplicated DNA. The digested DNA pool was then used to transform competent bacterial cells, and colonies containing replicated plasmid DNA were isolated. The sequence of plasmid DNA from 60 such clones was obtained using an ABI Prism 3700 DNA Analyzer (PerkinElmer Life Sciences) (see Table VI).
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RESULTS |
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Recovery of Autonomously Replicating SequencesAfter synthesis of oligonucleotides containing T3, T7, and M13 reverse primers bracketing the consensus sequence, the mixed pool of oligonucleotides was amplified by PCR using the primers and ligated into pCRscript using the SrfI restriction site. The ligation pool was used in transfection of HeLa cells and as a template in an in vitro DNA replication system using HeLa cell extracts as a source of replication proteins (11). (Previously, we have shown that in this in vitro replication system, initiation is site-specific and maps to the same site as in vivo (8, 11, 19).) To eliminate unreplicated DNA, the recovered DNA after transfection into HeLa cells or in vitro replication was digested with DpnI. Then, the pool of DNA products from both replication systems, presumably containing some replicated DpnI-resistant plasmid + consensus inserts, was used to transform competent bacteria and obtain bacterial clones of versions of the consensus sequence capable of autonomous replication. All bacterial clones recovered after selection with ampicillin were found to contain plasmid constructs as identified by agarose gel electrophoresis of plasmid DNA preparations. 17 independent clones were sequenced and shown to contain various versions of the consensus with appropriate flanking sequence. Table I summarizes the consensus sequences recovered. Within the 17 clones, there were found multiple representations of some versions of the consensus; the A3/4 version of the consensus sequence was represented 5 times in this group of 17 clones. When these 17 sequences were compiled to generate another consensus sequence, we derived the same 36-bp consensus used to generate the oligonucleotide mixture used in this study. Only 3 clones, A1, A5, and A39 (Table I), were uniquely represented in the group of 17. We next asked whether the assortment of the 17 sequences could be considered as different from randomly selected clones, i.e. without selection by replication in our in vitro system. We randomly picked and sequenced 8 clones transformed with an aliquot of the same ligation pool, but not subjected to replication in vitro. All 8 clones were found to be unique versions with no multiple representations of any single version. As shown in the Fischer's exact test of autonomously replicating sequences (Table II), the probability that the 17 independent clones recovered after replication in the in vitro system could be attributed to random sampling of the available plasmid + consensus insert versions is unlikely (p = 0.000153). In other words, the library from which autonomously replicating clones were obtained was not limited and did not have any apparent overrepresentation of the replicating clones.
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Occurrence of Homologs in Data Base SequencesA search for homologs in the GenBank data base revealed significant similarity to a number of sequences, including some in regions wherein origins of DNA replication have been mapped. None of the sequences within such regions were 100% similar to the 36-bp consensus. Homologs varied in similarity from 71 to 88% over 21 to 35 bp in initiation regions for c-myc (20), lamin B2 (21), NOA3 (22), -globin (23), IgMµ chain enhancer (24), heat shock protein 70 (25), the Chinese hamster ovary dhfr (26), and the rodent RPS14 (27). In vivo footprinting of the lamin B2 origin region revealed that an area of 70 bp was protected on one strand (28). More recently, a 79% homology match over 24 bp of the 36-bp consensus sequence was observed at the human lamin B2 origin site and was mapped to the 5'
3' strand at a predicted bidirectional start site, position 3933 (29).
Among the most interesting homologs were those present in sequences isolated and characterized as CpG islands (30). CpG islands are regions of about 1 kb that are GC-rich (65%) and occur in association with promoters of about 50% of all mammalian genes (31, 32). Replication origins have also been detected at several promoters, including those for the c-myc gene (20, 33, 34), the Hsp70 gene (25), the ppv1 gene at the 3'-end of lamin B2 (21), and rat aldolase B gene (35). Based upon this background, Delgado et al. (36) showed that CpG islands are initiation sites for both transcription and DNA replication. Over 36 bp, several sequences, identified as CpG islands (30), had 83 and 89% homology; and with the allowance of a single base gap over 35 bp, several CpG island sequences contained homologs of 100% similarity (Table III). A bidirectional origin of replication was mapped 3' to the chicken lysozyme locus within a CpG island (37). Analysis of the 600-bp region within which the initiation site resides showed a 70% homology to the consensus over 24 bp.
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Autonomous Replicating Activity of Consensus Versions Using the BrdUrd semiconservative replication assay, as described previously (6, 7, 38), we examined the ability of a number of plasmid clones for autonomously replicating activity after transfection into HeLa cells, bovine embryo kidney fibroblasts, and chicken embryo fibroblasts (Table IV). After one round of semiconservative replication, the DNA product should be of heavy-light (HL) hybrid density, whereas two or more rounds of replication should yield fully substituted DNA of heavy-heavy (HH) strands. Plasmids containing the A3/4 and A16 versions of the consensus sequence (for sequence, see Table I) exhibited efficient autonomous semiconservative replication (Fig. 1). For both plasmid clones shown, a peak of unreplicated (LL) DNA was recovered at the top of each gradient (Fig. 1A, region including fractions 2224; and Fig. 1B, region including fractions 1924). Additional peaks of replicated, HH DNA were also obtained, indicating two or more rounds of replication, respectively (Fig. 1, A and B), with some potential HL DNA in Fig. 1A. The linearity of each gradient was verified by measuring the refractive index of every other fraction (Fig. 1, A and B). As negative control, a plasmid vector (pCRscript) alone was transfected in separate flasks, for which only LL DNA was recovered (data not shown; examples of negative controls can also be found elsewhere in Refs. 6, 7, 12, 38, and in Fig. 4, A and C, below).
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Two clones of CpG islands (30), CP9 (HS14C3R locus) and 6K (HS8F7F locus), which contain versions of the consensus sequence, were also tested for autonomous replicating activity by the semiconservative BrdUrd incorporation assay after transfection into HeLa cells (Fig. 3). Only CP9 (Fig. 3A) was able to support autonomous replication after transfection into HeLa cells readily, with high amounts of both HH and HL replicated plasmid DNA, whereas clone 6K (Fig. 3B) was incapable of autonomous replication because all plasmid was recovered as unreplicated (LL).
We also tested whether versions of the consensus sequence might support autonomous replication of plasmid DNA after transfection into eukaryotic cells of other species. Bovine embryo fibroblasts and chicken fibroblast cells were transfected with plasmid DNA containing different versions of the consensus sequence (Fig. 4). A negative control plasmid (clone 30.4; Fig. 4A) and the CpG island clone 6K containing the consensus (Fig. 4B) were tested for autonomous replication activity in bovine embryo kidney fibroblasts. The 6K plasmid clone showed strong autonomous replication activity with high amounts of both HL and HH DNA being recovered (Fig. 4B) relative to plasmid clone 30.4 (Fig. 4A), in which the majority of DNA was recovered as unreplicated (LL). In Fig. 4, C and D, clone 30.4 and the CP9 consensus clone, respectively, were tested for autonomous replication activity in chicken embryo fibroblasts. Again, in these cells, clone 30.4 exhibited a very low (background) level of replication (Fig. 4C), whereas clone CP9 exhibited efficient autonomous replicating activity, with large amounts of HH and HL DNA being recovered (Fig. 4D). Finally, the autonomously replicating activity of clone A16 (see Table I) in chicken embryo fibroblasts (Fig. 5A) was compared with that of clone 6K (Fig. 5B). Although clone A16 was able to replicate autonomously in chicken embryo fibroblasts, the activity of clone 6K in these cells was very low.
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We next assessed the ability of the different versions of the consensus sequence (i.e. A3/4, 6K, A16, and CP9) to replicate in normal human cells (WI38 embryo lung fibroblasts) and in immortal murine fibroblasts (3T3 cells). For this, we used the DpnI resistance assay to detect plasmid DNA replicated in mammalian cells that lack a deoxyadenosine methylase (dam) gene, making the replicated DNA resistant to digestion with DpnI. After low molecular weight DNA preparations from Hirt lysates of transfected cells were digested with DpnI, the DNA was used to transform bacteria, as an indicator of autonomous replication potential as previously described (10). As shown in Fig. 6, both mouse 3T3 cells and normal human (WI38) cells, supported the replication of the consensus sequence variants, compared with a negative control plasmid, 30.4. The most efficient replication was observed with the A3/4 consensus version plasmid in both mouse and human cells. 6K also demonstrated autonomous replicating activity in 3T3 cells, but not in human cells, consistent with the results obtained after its transfection into HeLa cells, using the semiconservative assay for autonomous replication (Fig. 3B). A16 and CP9 also replicated autonomously in both mouse and human cells, albeit with much lower efficiency than A3/4 or 6K (Fig. 6); both A16 and CP9 replicated with higher efficiency in mouse 3T3 cells than in human (WI38) cells. Table III summarizes these results.
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Finally, a double-stranded oligonucleotide of 40 bp (TTTTTTTTTTCCAATGATTTGTAATATACATTTTATGACT), spanning the region inclusive of the lamin B2 origin and start site (29) with homology to the consensus sequence (see "Occurrence of Homologs in Data Base Sequences") was cloned into pBluescript II. This plasmid was then tested for its ability to support autonomous replication in HeLa cells by the DpnI resistance bacterial retransformation assay, as described previously (7, 9, 10). In preliminary experiments, the sequence inclusive of the lamin B2 start site (107 ± 36 colonies/plate, mean ± S.D. of three plates) was found to support autonomous replication as efficiently as did the 36-bp A3/4 consensus version cloned into pBluescript II (70 ± 17 colonies/plate; background using a plasmid without consensus or lamin B2 sequence was 7 ± 2 colonies/plate).
Stability of pYACneo Constructs Containing the A3/4 Consensus SequenceThe A3/4 version of the consensus sequence was subcloned from the pCRscript clone into the EcoRI restriction site of pYACneo. After transfection into HeLa cells, independent clones were selected with G418 and maintained continuously in culture in the presence of 400 µg/ml G418, as described previously (10). After >170 cell doublings, one of the clones was labeled with BrdUrd and then low molecular weight episomal DNA was recovered. The DNA was loaded onto a CsCl gradient, and fractions were collected, blotted, and hybridized with pYACneo containing the A3/4 insert. As shown in Fig. 7, there is an absence of the usual high amount of unreplicated (LL) DNA present in short term (23 days after transfection) assays. There are additional peaks of replicated, HL and HH DNA, indicative of continuing efficient semiconservative replication of this episome in the HeLa cells. As before, the linearity of the gradient was verified by measuring the refractive index of every other fraction (Fig. 7). As a negative control, the pYACneo vector alone was transfected and monitored in parallel in separate flasks, for which only LL DNA was recovered (data not shown). Table V summarizes the results; for comparison, the data from previous fluctuation tests of short mammalian origin sequences maintained as HeLa episomes are also shown. HeLa cells transfected with pYACneo alone yielded stable cell clones (three of three) that had integrated the plasmid into the genomic DNA. pYACneo was not observed to be maintained as an episome. As can be seen for all six independent clones, there was no integration of plasmid and the pYACneo + A3/4 construct (A3/4 in pYACneo) was maintained as an episome. Furthermore, the stability of the episome in the absence of selection was found to be 0.9/cell/generation compared with the nonepisomally maintained (integrated) plasmids that had a stability of 1.0/cell/generation. Low molecular weight episomal DNA was used to obtain bacterial transformants; the plasmid DNA recovered in three independent clones was tested with several different restriction enzymes to indicate any apparent rearrangements. For example, digests of DNA with AvaI and HindIII enzyme gave the predicted fragment size of DNA from each of three independent plasmid clones recovered from two of the six independent HeLa cell clones that contain only nonintegrated episomal pYACneo + A3/4 DNA (Fig. 8 and Table V).
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Autonomous Replication of Consensus Sequence Containing Plasmids in Drosophila CellsThe apparent activity of versions of the consensus sequence across many species, including the taxonomic classes, Mammalia and Avia (see Table IV), caused us to wonder whether versions of the consensus sequence might be active across phyla of Chordata and Arthropoda (e.g. Drosophila melanogaster, an invertebrate). Because homology (6673%) was detected in Drosophila DNA to the consensus sequence, we tested the ability of A3/4 to support autonomous replication across phyla of Chordata and Arthropoda (e.g. D. melanogaster, an invertebrate), by the semiconservative BrdUrd incorporation assay. After transfection of the A3/4 version of the consensus sequence cloned in pCRscript (pCRscript + A3/4) into Drosophila S2 cells, peaks of DNA near the HL and HH positions of the gradient were recovered (Fig. 9, open bars), indicative of autonomous replication, whereas the negative control plasmid, 30.4, was replication-negative (Fig. 9, solid bars). An unusual feature was the virtual absence (very low level) of input (LL) DNA recovered from either the (pCRscript + A3/4) or from clone 30.4 plasmids. Such a result suggests that in Drosophila cells the input plasmids that are not competent for replication were degraded rapidly.
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Preliminary Mutagenesis StudiesTo test further the potential of this consensus sequence in control of eukaryotic DNA replication, preliminary mutagenesis studies of a version of the consensus sequence were conducted. Random mutagenesis was performed on the A3/4 version of the consensus sequence, resulting in 52 changes that occurred in 38 variant clones: 2 gaps; 8 pyrimidine to pyrimidine changes; 26 pyrimidine to purine changes; 10 purine to purine changes; and 6 purine to pyrimidine changes. Only 5 bases (at positions 3, 13, 14, 16, and 22; asterisks in Table VI) of the 36 bases had no change that was detectable in any of the clones. A pool of equimolar amounts of plasmid DNA obtained from each of the 38 clones plus A3/4 was transfected into HeLa cells. Of the 38 variant clones, 10 clones (representing those sequences obtained from more than a single bacterial colony) were recovered as resistant to DpnI and having replicated in the HeLa cells. These clones were detected by sequencing of DpnI-resistant plasmids isolated from each of 60 bacterial colonies. These 10 mutated versions of A3/4 plus unmutated A3/4 were found among 47 bacterial colonies (Table VI), whereas 13 additional clones were found represented in only a single bacterial colony. Thus a total of 23 of the 38 variant clones were resistant to digestion by DpnI, indicating that they had replicated autonomously in HeLa cells.
For statistical analysis, a more stringent criteria of segregating the clones was used. The replicating, DpnI-resistant plasmids that were able to transform bacteria and were detected among 60 bacterial colonies 1) more than once or 2) not at all were compared with those that were 3) not detected as replicating or 4) detected in only a single bacterial colony of the 60. For those clones represented more than once among the 60 bacterial colonies containing plasmid, the probability (Fischer's exact test) that 1) the 20-bp region (position 322; see line in Table VI) in the 36-bp A3/4 sequence would be present in all of the 10 variant clones as unmutated and 2) that there would be 17 clones with mutations in the 20-bp region or 11 clones outside the regions that were not represented more than once or at all is p < 0.03. (Note that there are two clones, clones 2 and 7, included as unmutated in the 20-bp region because the mutations are permissive relative to the consensus sequence; see Table VI and footnotes.) Thus, the 20-bp region has been identified as a putative minimal sequence that appears to be necessary.
If the 20-bp internal sequence (322 in the 36-bp consensus sequence) is used for assessment of homology, the homology to CpG islands as shown in Table III improves in most cases, with many 100% homologies with no gaps (Table VII). There is only one case in which a gap is now detected (accession no. Z54973 [GenBank] ).
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For two regions mapped as initiation sites of DNA replication in c-myc, there is between 75 and 89% homology to the 20 bp over 18 bp to 20 bp (20, 34). The homology for the 20 bp to lamin B2 adjoins the initiation site in the lamin B2 locus (21, 29). The homology for the autonomously replicating sequence and origin known as NOA3 (12, 22) and heat shock protein 70 (25) is also shown in Table VII. An origin of DNA replication has been reported for the Chinese hamster ovary dhfr locus (26), and there are homologies of 89% present on each strand as located within an autonomously replicating fragment, X24 (8).
We have used two 20-bp duplexes, each placed separately into the EcoRI site of pBluescript, to test for autonomous replication ability in HeLa cells using the DpnI resistance assay and bacterial retransformation. Clone 20 is identical to the relevant 20 bp of A3/4 except in the last position in which there is a G instead of C. Clone 85 is identical to A3/4 except for inversion of the first 2 bases from TC in A3/4 to CT in the 20-mer clone (see Table VI). 20-mer clone 20 gave 85 ± 16 (S.D.); 20-mer clone 85 gave 54 ± 14; and A3/4 gave 63 ± 13 bacterial colonies/plate. (Background, pBluescript vector alone (21 ± 7), was subtracted from these values.) We also tested two examples of mutations in the internal 20-bp sequence of the A3/4 sequence which were not recovered as one of the replicating clones. Mutated clone A1 has a nonpermissive change from G to A at position 9 of the 36-bp consensus, and mutated clone C2 has a change from A3/4 of T to C at position 11 of the 36-bp consensus sequence. In autonomous replication experiments, A3/4 gave 41 ± 7 and clone 85 gave 42 ± 6, whereas mutated clone A1 and mutated clone C2 gave 0 ± 0 and 1 ± 1, respectively, demonstrating that these changes within the 20 bp of the consensus seemed to affect replication activity greatly. In other preliminary experiments, both 20-mer clones (clone 20 and clone 85) competed with the 36-bp A3/4 sequence for OBA/Ku86 binding (41, 42, 43) (data not shown).
Distribution of 20-Mer Consensus Sequence on Human ChromosomesThe distribution of the 20-mer consensus sequence over 1 Mb of continuous human genomic sequence for chromosomes 1, 20, 21, and 22 was examined using fuzznuc of the EMBOSS suite of software. The results shown in Table VIII were obtained for up to two through five mismatches (90 through 75% homology) allowed with no gaps. Under these conditions, two mismatches gave a range of 1951 homologs, whereas more mismatches rapidly increased the number of homologs to a maximum of 9,10112,597 for five mismatches, no gaps. The distribution on the DNA +/- strands was approximately equal. For the allowance of two mismatches and assuming an equal distribution, initiation sites would be spaced from 20 kb to
50 kb apart. However, as demonstrated in Fig. 10, the distribution is not equal and can vary from ≤1,000 bases to ≥200 kb. A comparison with the distribution of the Saccharomyces cerevisiae ARS core consensus sequence, WTTTATRTTTW, using fuzznuc with no mismatches for chromosomes IV, VII, XII, XV over the first 1 Mb of sequence as obtained from the Saccharomyces Genome Data base,3 indicated 25/23 (+/-), 20/23, 20/25, 17/19 homologs, respectively. The total of homologs, ranging from 36 to 48, compares favorably with the 20-mer consensus sequence on 1 Mb of human chromosomes.
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The homologs of the 20-mer sequence did not overlap any CpG islands (UCSC Genome Browser v17; >200-base length, >0.5 GC content, and the ratio of >0.60 for observed proportion of CG dinucleotides to the expected proportion on the basis of the GC content of the segment); that is, 0/13 in chromosome 1, 0/6 in chromosome 20, 0/1 in chromosome 21, 0/11 in chromosome 22. For -satellite sequence, only the 1 Mb of sequence at chromosome 22q11.1 contained an example of
-satellite that did overlap 3 homologs and corresponding to those with the 3 above the vertical bar in Fig. 10. Homologs (2 mismatches, no gaps), using BESTFIT of the GCG suite of software, of the 20-mer sequence to
-satellite and centromere sequence of the individual chromosomes were 4/6 in chromosome 1, 1/10 in chromosome 20, 4/10 in chromosome 21, and 1/10 in chromosome 22.
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DISCUSSION |
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A human origin binding activity (OBA) has been isolated using a minimal 186-bp fragment from the monkey autonomous replicating sequence ors8 (43). Homology to the consensus sequence was observed within a 59-bp fragment that was the most effective competitor for binding of OBA to the 186-bp minimal autonomous replicating sequence. We then showed that A3/4 (36 bp) consensus was as effective a competitor as the 59-bp fragment and used it to affinity purify OBA, which was identified as the Ku86 subunit of Ku antigen (42). Ku antigen is identical to a DNA-dependent ATPase isolated from HeLa cells (48), which had been reported previously to cofractionate with a 21 S multiprotein complex competent for DNA synthesis from HeLa cells (49) and is capable of interaction with a region containing the replication origin of lamin B2 (41). The finding that a version of the consensus sequence is an effective competitor for OBA/Ku86 binding to a minimal autonomously replicating sequence lends support to the functionality of at least some versions of the consensus sequence. More recently, we have demonstrated, using chromatin immunoprecipitation assays, that Ku is associated in vivo with mammalian origins of DNA replication including ors8 and ors12, in a cell cycle-specific fashion, namely at G1/S (50). Recently, we have also obtained DNase I footprints of OBA/Ku86 and recombinant Ku upon a plasmid fragment containing A3/4, including bases in positions of 13, 929, and 3236 (includes part of the 20-bp internal autonomously replicating sequence, positions 322); similarly, a footprint was obtained over the consensus homologous regions of ors8 (51). The isolation and identification of this origin binding activity, using A3/4 as an affinity purification step, and its in vivo association with origins of DNA replication, provide further supporting evidence consistent with A3/4 possessing origin activity in episomes as well as in vivo,in chromatin.
Various versions of the 36-bp consensus sequence were capable of autonomous replication after transfection into HeLa cells, as shown by the BrdUrd incorporation assay, leading to the production of both HL (hybrid density) and HH (fully substituted) DNA, diagnostic of semiconservative replication. The consensus served as an initiation site, as shown by the earliest labeled fragment method in a mammalian in vitro DNA replication system (11), which mapped the earliest incorporation of radiolabeled nucleotides to a minimal fragment of the plasmid containing the A3/4 version of the consensus. Two of the CpG island clones, CP9 and 6K, which contain versions of the consensus sequence, were also tested for autonomous replication activity, with one (CP9) demonstrating autonomous replicating activity in HeLa cells. However, analysis of autonomous replicating activity of the various versions of the 36-bp consensus and homologs present in the CpG island sequences in human, bovine, and chicken cells suggested that there may be species preference for subsets of the versions of the consensus and its homologs. For example, 6K clone was found to have activity in bovine cells, but not in human and chicken cells, whereas CP9 clone had significant activity in chicken and human cells. This apparent species preference may be associated with the initiator proteins involved in the recognition of the critical nucleotide sequence elements of the consensus and its homologs. Furthermore, the context in which the consensus sequences are present was varied from multiple cloning sites in pCRscript, and pBluescript to CpG islands (Table III) and to the EcoRI site of pYACneo. It was not possible in these experiments to establish fully what contribution, if any, context may play in the activity of consensus sequences.
Versions of the consensus sequence, particularly A3/4, were found to replicate in both normal and malignant human cells (WI38 and HeLa, respectively). A pYACneo construct containing A3/4 could be maintained exclusively as episomes under selection for long periods of time. After ≥170 cell doublings, we demonstrated the continuing autonomous replication of the episomes in HeLa cells. Episomes recovered did not indicate any rearrangements of the constructs introduced into HeLa cells. Removal of selective pressure demonstrated that the episome had a surprising stability of ≥0.9/cell/generation.
In anticipation of the next step in derivation of a minimal consensus core sequence for eukaryotic and mammalian DNA replication, preliminary mutagenesis studies were done in which a minimal 20-bp region is apparently correlated with the ability to replicate in HeLa cells. The distribution of this 20-mer consensus sequence over 1 Mb of human chromosomes is similar, quantitatively and qualitatively (relative proximity to each other) to the distribution of ARS sequence on S. cerevisiae chromosomes. However, it may be that specific elements or combination of bases involved in initiator protein or cooperating replicative proteins binding are still to be revealed, allowing further delimination of a minimal core consensus sequence. With functional testing and further mutagenesis, it should be possible to derive a minimal core consensus sequence. It appears likely that the 20-bp sequence might be required for control of autonomous replication. In a context related to its position with regard to other surrounding sequence, the associated sequence may play a role in regulation of replication origin activity at different times and in different cell types.
This consensus will provide for a similar advancement in understanding of regulation of DNA replication in higher eukaryotes, as the yeast ARS consensus did for DNA replication in yeast. With this greater definition will come a more rational approach to the development of compounds that affect DNA replication. This technology also has direct application to the development of nonviral vectors for gene transfer. Currently, wherein adenoviral gene delivery systems in particular and gene therapy in general are under close scrutiny because of adverse effects in gene therapy trials (52, 53), a consensus sequence of host cell composition, which can maintain DNA replication and expression of accompanying genes, provides a new opportunity for a gene delivery system of cellular (host) DNA origin.
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FOOTNOTES |
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¶ Recipient of an Fonds pour la Formation de Chercheurs et l'Aide à Recherche Centre Studentship and Canadian Institutes of Health Research doctoral research studentship.
Recipient of a Canadian Institutes of Health Research studentship.
To whom correspondence should be addressed: McGill Cancer Centre, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G 1Y6, Canada.
1 The abbreviations used are: ARS, autonomous replicating sequence; BrdUrd, bromodeoxyuridine; HH, heavy-heavy; HL, heavy-light; LL, light-light; OBA, origin binding activity; ors, origin-enriched sequence(s).
2 Further details of sequence and method to generate the consensus are available upon request. Nucleotide code: M = A or C; D = A, G, or T; W = A or T; K = G or T; S = C or G; B = C, G, or T; Y = C or T; R = A or G; H = A, C, or T; V = A, C, or G; N = A, C, G, or T.
3 K. Dolinski, R. Balakrishnan, K. R. Christie, M. C. Costanzo, S. S. Dwight, S. R. Engel, D. G. Fisk, J. E. Hirschman, E. L. Hong, L. Issel-Tarver, A. Sethuraman, C. L. Theesfeld, G. Binkley, C. Lane, M. Schroeder, S. Dong, S. Weng, R. Andrada, D. Botstein, and J. M. Cherry, ftp://genome-ftp.stanford.edu/pub/yeast/SacchDB/ March 11, 2003 (date of access).
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