Background Transmission of genetic information from one cell generation to the next requires the accurate duplication of the genome. Replication initiation is a well-conserved process determined in all eukaryotes by the binding of the pre-Replication Complex (ORC and MCM proteins) to replication origins. Despite the early success in budding yeast S.cearevisiae replication origins mapping, in mammals only few origins have been identified, because no consensus sequences have been found and epigenetic seem to be important for their selection. Objectives To study if and how genetic and epigenetic features can determine origin activity and how this process might be linked to gene expression, the identification and characterization of DNA sequences that serve as replication origins in mammals is crucial. Results & Discussion We have developed a novel strategy to identify human replication origins based on ultracentrifugation in equilibrium density gradient of sheared cross-linked chromatin. Our results show that known replication origins are enriched in high-density fractions, containing naked DNA. We then probed tiled oligonucleotide microarrays (Nimblegene Technology) containing the human genomic DNA from chromosome 19 with origin-rich naked DNA and with DNA purified from ChIP assays with antibodies directed against proteins of the pre-RC complex. The combined analysis of these two hybridizations allowed the identification of about 30 candidate replication origins. With an independent assay we confirmed that 80% of these regions are newly identified replication origins. Conclusions We found an excellent tool for high-throughput identification of human DNA replication origins, very useful for their characterization. References 1. Bell, S. and A. Dutta, DNA replication in eukaryotic cells. Annual Review Biochemistry, 2002. 71: p. 333-74. 2. Cook, P., The organization of replication and transcription. Science, 1999. 284. 3. Schwartz, Y., T. Kahn, and V. Pirrotta, Characteristic low density and shear sensitvityof cross-linked chromatin containing polycomb complexes. Molecular and Cellular Biology, 2005. 25(1): p. 432-439.
|Titolo:||IDENTIFICATION OF NEW HUMAN REPLICATION ORIGINS|
|Data di pubblicazione:||giu-2007|
|Citazione:||IDENTIFICATION OF NEW HUMAN REPLICATION ORIGINS / S. Banfi, G.I. Dellino, M.Cesaroni, S. Segalla, A. Brozzi, L. Luzi, P.G. Pelicci. ((Intervento presentato al convegno DMMC International Workshop Epigenetics : from mechanism to medicines tenutosi a Dublino nel 2007.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|