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Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences.

Valenzuela MS, Chen Y, Davis S, Yang F, Walker RL, Bilke S, Lueders J, Martin MM, Aladjem MI, Massion PP, Meltzer PS - PLoS ONE (2011)

Bottom Line: Our results suggest that the program for origin activation is largely conserved among different cell types.Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection.Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. mvalenzuela@mmc.edu

ABSTRACT

Background: Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.

Methodology/principal findings: We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7-1.5 kb obtained from the breast cancer cell line MCF-7 hybridized to a custom tiling array containing 50-60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5'ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.

Conclusions/significance: Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.

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Related in: MedlinePlus

Comparison of nascent DNA enrichment profiles obtained with MCF-7                            nascent DNA fractions of increasing size.(A) DNA from sucrose gradient fractions containing nascent DNA with a                            size range of, 0.7–1.5 kb (fraction 10–12); 1.5–3 Kb                            (fraction 18); and >3 kb (fraction 28) obtained from the same MCF-7                            preparation were hybridized to the tiling array. As a control, sonicated                            total DNA in the size range of 0.7–1.5 kb was utilized as both                            test and reference DNA (self-self). After hybridization, the                            test/reference ratios (black dots) were processed to produce a smoothed                            line (red line). The peaks represent sites where enrichment of nascent                            DNA (origins) occurred along a 90 kb region of chromosome 17 (B)                            Comparison of origin profiles along two (13 kb and 43 kb respectively)                            selected regions on chr17 (indicated as bars in Figure 1A) obtained from either array                            data (red line) or from real-time PCR data (black line). (C) Peak                            spacing densities derived from nascent DNA enrichment profiles for the                            three DNA size range preparations hybridized to the array. Peaks were                            detected using a Savitzky-Golay filter with a 500 bp moving window over                            the genomic regions covered by the array. The number of peaks detected                            in each of the preparations is indicated in parentheses.
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pone-0017308-g001: Comparison of nascent DNA enrichment profiles obtained with MCF-7 nascent DNA fractions of increasing size.(A) DNA from sucrose gradient fractions containing nascent DNA with a size range of, 0.7–1.5 kb (fraction 10–12); 1.5–3 Kb (fraction 18); and >3 kb (fraction 28) obtained from the same MCF-7 preparation were hybridized to the tiling array. As a control, sonicated total DNA in the size range of 0.7–1.5 kb was utilized as both test and reference DNA (self-self). After hybridization, the test/reference ratios (black dots) were processed to produce a smoothed line (red line). The peaks represent sites where enrichment of nascent DNA (origins) occurred along a 90 kb region of chromosome 17 (B) Comparison of origin profiles along two (13 kb and 43 kb respectively) selected regions on chr17 (indicated as bars in Figure 1A) obtained from either array data (red line) or from real-time PCR data (black line). (C) Peak spacing densities derived from nascent DNA enrichment profiles for the three DNA size range preparations hybridized to the array. Peaks were detected using a Savitzky-Golay filter with a 500 bp moving window over the genomic regions covered by the array. The number of peaks detected in each of the preparations is indicated in parentheses.

Mentions: Upon hybridization of the 0.7–1.5 kb nascent DNA (test DNA) and similarly size sheared total DNA (reference DNA) to the tiling array, we observed a strong short-range autocorrelation among neighboring probes (Fraction 10–12, Fig. 1A), which was absent in the input\input hybridization (self-self, Fig. 1A). These results suggested that the peak signals observed with the short DNA arose from the enrichment of regionally localized DNA sequences in our DNA preparation. If the pattern of peaks and troughs derived from short nascent DNA, as opposed to randomly broken DNA fragments, we predicted that the peak signals would diminish in fractions containing larger DNA fragments. To test this, we examined fractions containing nascent DNA in the ranges of 1.5–3 Kb, and ≥3 kb DNA (Fraction 18 and Fraction 28, respectively, Fig. 1A). As predicted, their profiles were both quantitative and qualitatively different from that of the fraction 10–12 DNA pool, yielding progressively fewer and broader peaks. We interpreted these results as indicative of enrichment for origins of DNA replication in the 0.7–1.5 kb fraction, that decreased with fragment size, since the ratios of signals emanating from test versus reference DNA were greatest in the short nascent DNA and declined in fractions with larger fragments.


Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences.

Valenzuela MS, Chen Y, Davis S, Yang F, Walker RL, Bilke S, Lueders J, Martin MM, Aladjem MI, Massion PP, Meltzer PS - PLoS ONE (2011)

Comparison of nascent DNA enrichment profiles obtained with MCF-7                            nascent DNA fractions of increasing size.(A) DNA from sucrose gradient fractions containing nascent DNA with a                            size range of, 0.7–1.5 kb (fraction 10–12); 1.5–3 Kb                            (fraction 18); and >3 kb (fraction 28) obtained from the same MCF-7                            preparation were hybridized to the tiling array. As a control, sonicated                            total DNA in the size range of 0.7–1.5 kb was utilized as both                            test and reference DNA (self-self). After hybridization, the                            test/reference ratios (black dots) were processed to produce a smoothed                            line (red line). The peaks represent sites where enrichment of nascent                            DNA (origins) occurred along a 90 kb region of chromosome 17 (B)                            Comparison of origin profiles along two (13 kb and 43 kb respectively)                            selected regions on chr17 (indicated as bars in Figure 1A) obtained from either array                            data (red line) or from real-time PCR data (black line). (C) Peak                            spacing densities derived from nascent DNA enrichment profiles for the                            three DNA size range preparations hybridized to the array. Peaks were                            detected using a Savitzky-Golay filter with a 500 bp moving window over                            the genomic regions covered by the array. The number of peaks detected                            in each of the preparations is indicated in parentheses.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3094316&req=5

pone-0017308-g001: Comparison of nascent DNA enrichment profiles obtained with MCF-7 nascent DNA fractions of increasing size.(A) DNA from sucrose gradient fractions containing nascent DNA with a size range of, 0.7–1.5 kb (fraction 10–12); 1.5–3 Kb (fraction 18); and >3 kb (fraction 28) obtained from the same MCF-7 preparation were hybridized to the tiling array. As a control, sonicated total DNA in the size range of 0.7–1.5 kb was utilized as both test and reference DNA (self-self). After hybridization, the test/reference ratios (black dots) were processed to produce a smoothed line (red line). The peaks represent sites where enrichment of nascent DNA (origins) occurred along a 90 kb region of chromosome 17 (B) Comparison of origin profiles along two (13 kb and 43 kb respectively) selected regions on chr17 (indicated as bars in Figure 1A) obtained from either array data (red line) or from real-time PCR data (black line). (C) Peak spacing densities derived from nascent DNA enrichment profiles for the three DNA size range preparations hybridized to the array. Peaks were detected using a Savitzky-Golay filter with a 500 bp moving window over the genomic regions covered by the array. The number of peaks detected in each of the preparations is indicated in parentheses.
Mentions: Upon hybridization of the 0.7–1.5 kb nascent DNA (test DNA) and similarly size sheared total DNA (reference DNA) to the tiling array, we observed a strong short-range autocorrelation among neighboring probes (Fraction 10–12, Fig. 1A), which was absent in the input\input hybridization (self-self, Fig. 1A). These results suggested that the peak signals observed with the short DNA arose from the enrichment of regionally localized DNA sequences in our DNA preparation. If the pattern of peaks and troughs derived from short nascent DNA, as opposed to randomly broken DNA fragments, we predicted that the peak signals would diminish in fractions containing larger DNA fragments. To test this, we examined fractions containing nascent DNA in the ranges of 1.5–3 Kb, and ≥3 kb DNA (Fraction 18 and Fraction 28, respectively, Fig. 1A). As predicted, their profiles were both quantitative and qualitatively different from that of the fraction 10–12 DNA pool, yielding progressively fewer and broader peaks. We interpreted these results as indicative of enrichment for origins of DNA replication in the 0.7–1.5 kb fraction, that decreased with fragment size, since the ratios of signals emanating from test versus reference DNA were greatest in the short nascent DNA and declined in fractions with larger fragments.

Bottom Line: Our results suggest that the program for origin activation is largely conserved among different cell types.Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection.Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. mvalenzuela@mmc.edu

ABSTRACT

Background: Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.

Methodology/principal findings: We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7-1.5 kb obtained from the breast cancer cell line MCF-7 hybridized to a custom tiling array containing 50-60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5'ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.

Conclusions/significance: Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.

Show MeSH
Related in: MedlinePlus