Genome-wide sequence and functional analysis of early replicating DNA in normal human fibroblasts
© Cohen et al; licensee BioMed Central Ltd. 2006
Received: 11 October 2006
Accepted: 29 November 2006
Published: 29 November 2006
The replication of mammalian genomic DNA during the S phase is a highly coordinated process that occurs in a programmed manner. Recent studies have begun to elucidate the pattern of replication timing on a genomic scale. Using a combination of experimental and computational techniques, we identified a genome-wide set of the earliest replicating sequences. This was accomplished by first creating a cosmid library containing DNA enriched in sequences that replicate early in the S phase of normal human fibroblasts. Clone ends were then sequenced and aligned to the human genome.
By clustering adjacent or overlapping early replicating clones, we identified 1759 "islands" averaging 100 kb in length, allowing us to perform the most detailed analysis to date of DNA characteristics and genes contained within early replicating DNA. Islands are enriched in open chromatin, transcription related elements, and Alu repetitive elements, with an underrepresentation of LINE elements. In addition, we found a paucity of LTR retroposons, DNA transposon sequences, and an enrichment in all classes of tandem repeats, except for dinucleotides.
An analysis of genes associated with islands revealed that nearly half of all genes in the WNT family, and a number of genes in the base excision repair pathway, including four of ten DNA glycosylases, were associated with island sequences. Also, we found an overrepresentation of members of apoptosis-associated genes in very early replicating sequences from both fibroblast and lymphoblastoid cells. These data suggest that there is a temporal pattern of replication for some functionally related genes.
A highly organized and strictly controlled process is necessary to accurately replicate the six billion base pairs of DNA that are tightly packaged within the confined volume of the human diploid nucleus. In order to accomplish this task, DNA replication is initiated at distinct sites in the genome as cells enter the S phase . The regulation of initiation is important because once the S phase begins, a cascade of events results in the successive activation of new replication clusters in a temporally and spatially ordered manner [2, 3]. The order of activation of the estimated 30,000 replicons in the human genome  is maintained through successive cell cycles  and is tissue specific [5, 6]. It is not yet fully understood how this sequential firing of replicons, resulting in the orderly progression through S phase, is regulated in human cells, but disruption of this process can have far reaching consequences that include acquisition of genetic instability leading to cancer.
The distribution of early and late replicating DNA is seen cytogenetically when metaphase chromosomes are Giemsa banded. It has been known for some time that Giemsa-negative or reverse (R) bands replicate early in S phase while Giemsa-positive (G) bands replicate late [7–9]. While the exact molecular basis for chromosomal banding is not understood, it has been proposed to be related to differences in chromatin condensation , the arrangement of scaffold-loop structures  or differences in GC content between neighboring regions . Indeed, R bands were reported to have a higher GC content  and have a higher density of genes [13, 14] and CpG islands  than G bands. Short and long interspersed nuclear elements (SINE and LINE, respectively) were also found to be unevenly distributed; a higher SINE frequency was found in R bands while LINE elements were disproportionately found in G bands [16, 17].
The distribution of the above-mentioned sequence features of early replicating DNA in the giemsa-negative bands (R-bands) were determined using cytogenetic methods. More recently, studies by Woodfine et al. [18, 19] confirmed the cytogenetic data, utilizing a microarray approach. Microarrays containing human genomic sequences were used for comparative hybridization of DNA isolated from S phase and G1 cells. Using this methodology, they found a positive correlation between early replication and CpG islands, GC content, expressed genes, and Alu repeats (a member of the SINE class of repeats). A negative correlation was found with LINE elements . White et al.  who found enrichment of transcriptionally active (but non-protein encoding) regions in early replicating sequences from chromosome 22 used a similar approach. Jeon et al.  investigated replication timing on chromosome 21 and 22 using high density genome-tiling arrays. Gene density, exon density, and gene expression were all highly correlated with early replication in their study. They hypothesized that an open chromatin environment, as reflected by high exon density separates early replicating DNA from that replicating later. In another study, Gilbert et al.  separated compact and open chromatin fibers and studied their genomic distribution using microarrays containing a sub-set of genomic sequences. They found a positive correlation between open chromatin and early replication according to results from low-density genome-wide arrays. This correlation disappears however, when comparing chromatin structure data and time of replication from high-density arrays for chromosome 22.
We have taken a different approach to study characteristics of DNA that replicates early in the S phase. We reported previously the construction of a cosmid library enriched in early replicating sequences from normal human fibroblasts . In the present study, we end-sequenced and mapped clones from this library, identifying "islands" where clones were adjacent or overlapping. We then verified that our island sequences exhibited the features of early replicating DNA reported by others (i.e., high GC, Alu, and gene content, and open chromatin structure) as well as active promoters. Also, we determined more precisely the time of replication for selected islands and compared them to data reported by Woodfine et al. [18, 19]. We performed a more extensive analysis of repetitive sequences in early replicating DNA, including long terminal repeats (LTRs), DNA transposons, and inverted, tandem, and simple repeats. We also identified genes associated with islands of early replicating sequences and found an enrichment in WNT genes and DNA glycosylases and, using the GOstat program, an overrepresentation of genes involved in apoptosis. Finally, we analyzed genes that overlapped markers tested by Woodfine et al. [18, 19] for replication timing and found that apoptotic genes also replicate very early in lymphoblastoid cells.
Results and discussion
Synchronization of normal human fibroblasts and library construction
The construction of a subgenomic cosmid library enriched in early replicating sequences has been previously described . Briefly, normal human fibroblasts (NHF1) obtained in this laboratory from neonatal foreskin  were synchronized to early in the S phase by a combination of confluence arrest, followed by replating at lower cell density in the presence of aphidicolin. The DNA polymerase inhibitor aphidicolin allows for the initiation of DNA replication, but dramatically slows replication fork progression [25, 26] without altering the order of gene replication . Bromodeoxyuridine (BrdUrd) was added to aphidicolin-containing medium to label DNA replicated as cells entered the S phase. Nuclear DNA purified from cells harvested 24 h after replating was partially digested with Sau 3AI, and hybrid-density DNA was separated in CsCl gradients. The purified early-replicating DNA was cloned into the sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones).
Identification of islands
Distribution of islands.
Mean Replication Timing Ratio2
It has been previously shown in mammalian cells that chromosomal bands that do not stain with Giemsa tend to replicate early while bands that do stain replicate late [7–9]. Using the estimated chromosomal band positions available in the UCSC Genome Browser , the percentage of early replicating island DNA in each band class was determined. There is a significant enrichment (p < 0.001, χ2 test) in G-negative bands with ~59% of island DNA in this band class as compared to 45% of total sequenced genomic DNA in these bands. In contrast, only 17% of islands were found in the two darkest staining and heterochromatic late-replicating bands, while nearly 33% of genomic DNA is contained within these bands. When we examined those markers used by Woodfine et al. [18, 19] that comprise the earliest replicating 25% of markers tested (markers with replication timing ratios > 1.75), we found that ~70% were located in G-negative bands. From these studies with higher resolving power than previous cytogenetic analyses, we concluded that while early replicating sequences are found predominantly in G-negative bands, a sizeable portion is not.
Further validation of replication timing using synchronized normal human fibroblasts
PCR markers analyzed and their replication timing.
Primer set name
Replication time in hrs1(fibroblasts)
Woodfine replication timing ratio2
We tested nine markers from five islands that overlap with sequences that have Woodfine replication timing ratios of 1.72 and higher [18, 19]. Seven out of nine of these markers replicated in the first hour of S phase, the other two replicated in the second hour indicating that there was good agreement in replication time for fibroblasts and lymphoblastoid cells in the earliest replicating sequences. White et al. , who compared their fibroblast replication timing on chromosome 22 to the Woodfine et al. data , found similar results. Also, Janoueix-Lerosey et al. , who studied replication timing in neuroblastoma cells found that 60% of early replicating sequences also replicated early in lymphoblastoid cells.
Although our library is enriched in early replicating DNA as mentioned previously, it does contain a small fraction of middle and late replicating sequences. For example, we used two PCR primer sets to test two points in an island that overlaps a Woodfine marker with a replication ratio of 1.51. In our assay, we found that this island replicates between 4.6 and 4.8 hrs, just past the middle of S phase. Another island, also tested for replication timing, was one of the few that were very late replicating, with a replication time of 5.6 and 5.5 hrs and overlapping markers with Woodfine replication ratio of 1.36 and 1.25. While these markers correlated well with data from lymphoblastoid cells, this did not hold true for all markers tested in fibroblasts. For example, clone island chr22.1110 overlaps markers with Woodfine replication ratios of 1.55 and 1.55, but testing of two PCR markers in this island indicated that in fibroblasts it replicated in the first hour, not in the middle of S phase. Additionally, PCR marker chr6.1395C, which is located in a region with a replication timing ratio of 1.58, replicates at 5.4 hrs, as do primer sets A and B from that same island that have Woodfine replication ratios of 1.36 and 1.25. These differences in timing might be the result of differences in cell type and could reflect differences in gene expression and/or chromatin condensation  between lymphoblastoid and fibroblast cells.
Distribution of repetitive elements in early replicating DNA
Genomic features significantly different in islands compared with whole genomic DNA.
Islands that overlap with replication timing markers1
Islands with a ≥ 1.65-timing ratio1
Islands with a ≥ 1.75-timing ratio1
Islands with a ≥ 1.85-timing ratio1
As mentioned above, tandem repeats, which included micro- and mini-satellites, were found enriched in islands. It has been established previously that tandem repeats are distributed in a nonrandom manner, with most being found in non-coding DNA and in both intergenic regions and introns . This is probably due, with the exception of tri- and hexa-nucleotide repeats, to selection against frameshift mutations . An excess of tandem repeats in early replicating DNA however, has not been reported previously. Some of this enrichment may be linked to the other repetitive elements that are enriched in early replicating sequences. For example, Alu repeats often contain microsatellite-like regions at their 3' ends . Since we found an enrichment of Alu sequences in islands, this could explain some of the enrichment of certain types of A-rich microsatellite structures. To determine whether A-rich microsatellites were responsible for the enrichment, we looked at the distribution of individual classes of tandem repeats in islands. This included mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide repeats, as well those with a periodicity of greater than six bases [see Additional file 1]. We found that all classes of tandem repeats, except di-nucleotides, were present at a higher level in islands than in the genome as a whole. Di-nucleotide repeats were actually found to be slightly depleted in islands. It is not clear why di-nucleotide repeats do not follow the same early replicating trend as other tandem repeats. Changes in the length of and presumably the establishment of microsatellites are thought to be caused primarily by DNA slippage during replication . This mechanism would involve increases in the length of short, and decreases in the length of long tandem repeats . Most of these changes are corrected by the mismatch repair pathway . It is possible that there are differences in the process that causes slippage mutations , or the ability to repair, the different classes of tandem repeats. These differences would presumably be influenced by the genomic features associated with early replicating sequences.
Open chromatin in early replicating regions
Using the same 1 Mb clone microarray as Woodfine et al. , Gilbert et al.  determined ratios for the enrichment of open (positive) regions of chromatin for each marker. Sites that were depleted in open chromatin sequences were considered to be regions of more compact (negative) chromatin. Lymphoblastoid cells were used in the Gilbert et al.  study and chromatin conformation most certainly differs between fibroblasts and lymphoblastoid cells in select regions. Nonetheless, we hypothesized that the overall chromatin conformation should be similar in both cell types. Gilbert et al.  previously reported a good correlation between open chromatin and early replication, based on the replication timing results of the 1 Mb array reported by Woodfine et al. . They note, however, that except in the regions with the highest enrichment in open chromatin, this correlation diminishes when chromatin data is compared to the high-resolution replication data on 22q. Gilbert et al.  suggested that, since there were a multitude of sites that were not enriched in open chromatin but were nevertheless early replicating, the two physiological characteristics (open chromatin and early replication) were not functionally related.
Chromatin structure and replication timing. Chromatin ratios were determined by Gilbert et al. (2004).
Average Chromatin Ratio
Regions of Open Chromatin
Islands with a ≥ 1.65-timing ratio 1
Islands with a ≥ 1.75-timing ratio 1
Islands with a ≥ 1.85-timing ratio 1
We also investigated regions of 22q where a total of 28 islands overlapped markers from the high-resolution array where the Woodfine et al.  data suggested an early replication time (replication ratio ≥ 1.75) and where chromatin data was also available . Since our islands average 100 kb in length and the spacing of clones on this array averages 70 kb, it is not unusual for an island to overlap more than one array marker. Together, the 28 islands overlapped 20 markers found to be in a more closed chromatin state (log2 open < 0) and 26 markers in an open state (log2 open > 0). Based on this data using markers from the high-resolution array, there is clearly little correlation between early replication and open chromatin as noted by Gilbert et al. . However, this lack of correlation could be attributed to a difference in the cell types, and thus chromatin states, at these locations in lymphoblastoid and fibroblast cells. Our data from chromosome 22 indicates another possible explanation. We found that 20 of the 28 islands (71%) overlapped at least one marker with log2 open > 0, but some also overlapped an additional marker with log2 open < 0. An example of this type of distribution occurred with island chr22.1090, which was defined by 6 separate early replicating clones, is 140 kb in length, and overlapped three arrayed markers with log2 open chromatin ratios of -0.538, 0.435, and 0.951. It is possible in these instances that an origin of replication that fires early in the S phase may be contained in the region of open chromatin, and fork progression from this origin extends into the nearby regions of more compact chromatin. Identification of replication origins and replicon boundaries in these regions will help to determine if this is indeed the case.
Transcription and early replication
Islands were evaluated for features related to transcription. Islands had a higher gene density, and were enriched in transcribed and translated sequences as compared to the whole genome (Table 3). A correlation between early replication and actively transcribed regions has been previously established [19, 42]. It is, therefore, not surprising that CpG islands, CpG content, and GC content are also found at higher densities in early replicating DNA, as each of these parameters are related to gene density. Likewise, we found that islands had greater amounts of evolutionarily conserved elements (; [see Additional file 2]) that are also predominately associated with gene sequences.
In a recent report, Kim et al.  evaluated the transcriptional status of promoters in human fibroblasts (IMR90 cells) by analyzing these sequences for the binding of the RNA polymerase II preinitiation complex. Here, we evaluated the distribution of active promoters  in early replicating DNA islands. As expected, active promoter density is greater in the islands as compared to the genome as a whole. Again, differences in feature content are even greater when considering only the subset of islands that overlap markers tested by Woodfine et al.  with replication timing ratios of ≥ 1.65 (Table 3).
Functional analysis of early replicating genes
Although it has been known that expressed genes, especially house-keeping genes, replicate predominately in the first half of S phase , these studies have not defined with great accuracy how early in S phase they replicate nor which genes are replicated in successive cohorts. In the present study, we were able to examine this question with greater precision. We looked at genes associated with islands and found that nine (47%) of the 19 WNT genes (wingless-type MMTV integration site family) were present. The nine island-associated WNT genes were located in eight islands found on eight different chromosomes. This family of genes is involved in stem cell regulation, wound healing in dermal fibroblasts, and is associated with cancer in many tissue types . Islands were enriched in genes associated with DNA repair, especially those related to the base excision repair pathway [46, 47]. These include four of ten known human DNA glycosylases (MBD4, NTH1, MPG and NEIL2), and several genes involved in the repair of apurinic/apyrmidinic sites (XRCC1, POLB, POLL, PARP1, FEN1, and RPA3). MGMT, whose gene product directly repairs O6-methylguanines, is also associated with a library island. A complete list of genes associated with library islands can be found in Additional file 3.
GOstat results. Representation of different GO functional categories as determined by the GOstat program .
Woodfine replication markers1
Apoptosis (programmed cell death)
Frizzled-2 (WNT) signaling pathway
Intracellular ligand-gated ion channel activity
Transmembrane receptor protein tyrosine kinase activity
Transmembrane receptor protein tyrosine phosphatase activity
G-protein coupled receptor activity
Rhodopsin-like receptor activity
G-protein coupled receptor protein signaling pathway
Olfactory receptor activity
Sensory perception of smell
Proteasome complex (sensu Eukaryota)
Cation-transporting ATPase activity
Positive regulation of signal transduction
Intracellular protein transport
Receptor signaling protein activity
Regulation of transcription factor activity
Positive regulation of cellular process
Intracellular membrane-bound organelle
Cell surface receptor linked signal transduction
Transmembrane receptor activity
Antigen processing endogenous antigen via MHC class I2
MHC class I protein complex2
MHC class I receptor activity2
Calcium ion binding
Integral to membrane
Phosphoric ester hydrolase activity
Organismal physiological process
Glutamate signaling pathway
Sensory perception of taste
Positive regulation of cellular metabolism
Our previous studies showed that cells in the earliest part of the S phase of the cell cycle are most vulnerable to malignant transformation by chemical carcinogens [52–54]. We hypothesized that the vulnerability of cells at the start of the S phase to carcinogen-induced changes that lead to cancer is due to the increased probability of mutating a relevant genetic target (whether a cancer gene or a regulatory region) during its replication at the time of carcinogen treatment early in the S phase. The finding that apoptotic, WNT genes, and DNA repair genes replicate in this compartment of the S phase, therefore, provides novel information on possible targets for carcinogenesis that are consistent with previous reports for common alterations in cancer.
We also examined the distribution of gene ontology categories for replication timing data reported for lymphoblastoid cells by Woodfine et al. [18, 19]. We found that markers used in these studies [18, 19] overlapped with ~9000 genes in the Known Gene dataset. For GOstat analysis, markers tested by Woodfine et al. [18, 19] were separated into three bins based on replication timing ratios: > 1.75 (very early; 3213 genes), 1.5–1.75 (early; 2698 genes), < 1.5 (late; 3093 genes). Genes that mapped to each bin were then compared to the complete set of ~9000 genes. Results for over and underrepresented functional categories for each replication-timing bin are listed in Table 5 along with the GOstat results for islands. For GO hierarchies where multiple ontologies were overrepresented, the most specific member was listed. Complete results for GOstat analysis of islands and Woodfine replication markers can be found in Additional file 4. It should be noted that there were several functional categories that were overrepresented in the original set of genes that overlapped with Woodfine replication markers (Table 5). These categories were associated primarily with the major histocompatability complex (MHC) that is found on chromosome 6 where the replication timing markers were placed at high density as described previously .
As shown in Table 5, there are some overrepresented categories that are found in island DNA but not in any of the bins of replication markers tested in lymphoblastoid cells. One reason for this may simply be the position of replication markers tested by Woodfine et al. [18, 19] since these markers were not randomly distributed throughout the genome. For example, only four of the 19 WNT genes overlap with these markers, which would make it difficult to determine whether they were overrepresented in any replication timing bin. It is also possible that the way that we partitioned these markers into bins obscured some GOstat results.
Temporal replication patterns for functionally related genes have been shown previously for the β-globin  and immunoglobulin heavy-chain locus gene clusters  but not for genes dispersed throughout the genome. The GOstat results in Table 5 give us an indication that there are temporal patterns of replication for some functionally related genes. Apoptotic genes for example, are overrepresented in both islands and the earliest replicating fraction from lymphoblastoid cells. This finding leads us to believe that genes involved in apoptosis represent a subset of genes that have been selected to replicate very early in the S phase. Another example of a functional group of genes that replicate at about the same time are genes involved in nucleosome assembly. These genes replicate predominately in the second quarter of S phase based on replication timing ratios reported by Woodfine et al. [18, 19]. This time of replication overlaps with the time when histone genes are being actively transcribed; histone transcription levels increase at the onset of S phase but reach their peak at about mid way through S phase .
In summary, in this work we present a genome-wide method for the identification of sequences that replicate early in the S phase. This method relied on the isolation and cloning of early replicating sequences followed by the end-sequencing and mapping of these clones to the nearly complete human genome sequence. This allowed us to evaluate regions with overlapping clones that are similar in size to an average replicon and therefore should replicate at the same time in S phase. This method has the advantage over microarray studies of replication timing in that there should be less contamination from adjacent sequences when trying to identify genomic features that correlate with time of replication. We found that for the earliest replicating sequences in normal human fibroblasts, there is a positive correlation with open chromatin, gene and exon density, and active promoters. Based on our comparison of replication timing in fibroblast and lymphoblastoid cells, these regions are more likely to replicate at the same time in multiple cell types than sequences that replicate at other times in the S phase. In addition, there are a few subsets of functionally related genes, including those for apoptosis and WNT signaling, that are replicated at this time. All of these data suggest that the chromatin structure/gene composition of this compartment of the S phase is conserved and may be important for the maintenance of cell stability and if defective may confer genetic instability. We are currently in the process of identifying origins of replication and replicon boundaries in some of these very early replicating regions in order to understand further the processes that orchestrate the order of replication at the beginning of the S phase.
NHF1 cells were synchronized and incubated in BrdUrd for 24 hrs in the presence of aphidicolin as described above. After collection by trypsinization, cells were washed and resuspended in cold saline, fixed in 67% ethanol, then stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ) as described . Flow cytometric analysis was done on a FACscan (Becton Dickinson) and the number of S phase cells was quantified using WinMDI program (Joseph Trotter, Scripps laboratory, ).
End sequencing of library clones
Cosmid DNA template was purified in 96-well format using LigoChem ProPreps, with ProCipitate, a synthetically engineered protein-binding polyelectrolyte (modification of method described in Kelley et al. ). Cycle sequencing was performed using ABI Big Dye Terminator cycle sequencing kits and reactions were run on ABI 3700 or ABI 3730 sequencers.
Mapping of paired cosmid clone ends to the human genome sequence
Reads were trimmed to minimize those of low quality (Phred score < 20) and to remove vector sequences prior to their alignment to the genome. Trimmed sequences were aligned to the NCBI build 35 (April 2004) sequence assembly using BLAT  with the default parameters except ooc = 11. ooc and -stepSize = 5. Resulting alignments were filtered to retain only the best alignments, requiring at minimum 80% of the trimmed sequence aligning with at least 90% base pair identity. Alignments from the 5' and 3' ends of the same cosmid clone were paired when these alignments were on the same chromosome, in the proper orientation, at a distance of at least 30 kb and no more than 50 kb from each other, consistent with sizing that is expected with cosmid cloning. These paired ends delimit the full extent of the clone in the genome sequence. Unpaired end sequences, where the pairing end sequence was either not available, could not be aligned, or was not a correct distance or orientation, were also retained as "orphan" ends. Only clones uniquely placed by paired ends or an orphan end were considered for further analysis. In addition, clones that mapped to nearly the same location as another clone and originated from an adjacent well in the 96-well plates used for sequencing, were removed as probable contaminants.
Determination of early replicating islands
Positions of early replicating clones were merged to create "islands" of early replication. Only clones within 100 kb of each other were merged, and the resulting island could be no longer than 175 kb. At least two clones were required to form an island.
Extraction of genomic features
Additional file 2 lists all genomic features considered in this analysis. Except for gene and promoter counts, chromosome bands, and chromatin status, the percentage of each feature was determined for each specified genomic region. These percentages were normalized to a 100-kb window to allow for comparison. Chromatin status was considered only for those genomic regions that overlapped a clone assayed in Gilbert et al. .
Determination of features significantly different in two datasets
For a feature represented by real-valued numbers for each genomic region coming from one of two distinct sets, the non-parametric Mann-Whitney/Wilcoxson ranksum test was used to calculate z-scores, providing a measure of the difference of the content of that feature in the two sets. To determine whether this difference was significant, 10,000 balanced permutations tests  were performed. Those features where no more than one of the 10,000 permutations had greater z-scores were considered significantly different in the two sets of sequence regions.
Cell cultures, synchronization of normal human fibroblasts and isolation of replicating DNA
NHF1-hTERT cells, the immortalized cell line derived from NHF1 cells by ectopic expression of the catalytic subunit of telomerase , were used for these studies. NHF1-hTERT cells were grown in Dulbecco's modified essential medium containing 2X the concentration of MEM non-essential amino acids (GIBCO-BRL, Grand Island, NY). Growth media was supplemented with 2 mM L-glutamine (GIBCO-BRL) and 10% fetal bovine serum (HyClone Laboratories, Inc., Logan UT).
Cells uniformly labeled with 14C-thymidine were synchronized by a combination of confluence arrest, followed by replating at lower cell density and treatment with aphidicolin for 24 hr. After removal of the inhibitor, the cells progress through the S phase as a synchronous cohort. The synchronized cells were labeled with BrdUrd and 3H-thymidine during sequential 1-h periods of the S phase and the replicated, hybrid-density DNA was fractionated by CsCl gradient centrifugation [32, 62]. After dialysis and concentration, the total amount of replicated DNA in the samples was determined from the specific activity of 14C-labeled DNA.
Determination of replication timing in Brdu-labeled nascent DNA
DNA replicated in different windows of the S phase was tested for the relative abundance of selected genetic markers (Table 2). PCR primers were designed using Primer3  or Prime (GCG Version 11.0, Accelrys Inc., San Diego, CA). PCR was performed using the OmnE and PCR Express thermocyclers (Thermo Electron Corporation, Waltham, MA) with Thermo-Start DNA polymerase (ABGene inc., Rochester, NY). PCR reaction conditions and, capture and analysis of digital gel images were described previously . Equal amounts of replicating DNA from samples representing seven hrs of the S phase were used as template for PCR reactions. In order to obtain quantitative results, it was necessary to carry out the PCR reactions under non-saturating conditions, and to construct a standard curve with genomic DNA of the same size as the tested DNA [31, 32]. DNA used for these PCR standard curves consisted of purified human genomic DNA, which was sheared 10 times through a 21-gauge needle, in the same way as the BrdU-labeled DNA prior to CsCl gradient centrifugation . Table 2 lists data from all markers tested. A numerical value approximating the time of replication was determined for each PCR marker tested using a previously described weighted average method . Briefly, a synchronized population of cells travels as a cohort through the S phase with the broadness of the peak reflecting the degree of synchrony. Therefore, it is necessary to consider more than one hourly replication fraction when calculating replication time. In order to determine the replication timing for each PCR marker, we first normalized by assigning the value of one to the highest sample and scaling all the other measurements to their corresponding fraction. Then, we included in our weighted average analysis the peak replication fraction and any other fraction that had > = to 50% of the peak value. This method while not producing an exact replication time does provide a good estimate, and allows for comparison among markers.
short interspersed repeat element
long interspersed repeat element
long terminal repeats
normal human fibroblasts
mammalian-wide interspersed repeats
endogenous retrovirus 1
endogenous retrovirus L
wingless-type MMTV integration site family.
This work was support by NIH grants CA084493 and ES09112. We thank the DOE Joint Genome Institute for the cosmid end-sequencing.
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