title: Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences.
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.
  
  
  RESULTS
  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
  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.
authors: Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. mvalenzuela@mmc.edu
authors: Manuel S
authors: MS
authors: Valenzuela
authors: Yidong
authors: Y
authors: Chen
authors: Sean
authors: S
authors: Davis
authors: Fan
authors: F
authors: Yang
authors: Robert L
authors: RL
authors: Walker
authors: Sven
authors: S
authors: Bilke
authors: John
authors: J
authors: Lueders
authors: Melvenia M
authors: MM
authors: Martin
authors: Mirit I
authors: MI
authors: Aladjem
authors: Pierre P
authors: PP
authors: Massion
authors: Paul S
authors: PS
authors: Meltzer
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