Abstract
BackgroundThe process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells.ResultsWe have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC) in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5) and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin.ConclusionTopoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.
Highlights
The process of DNA replication requires the separation of complementary DNA strands
The data reveal that only a small fraction of total chromatin-bound topoisomerases are located in the neighborhood of origin recognition complex (ORC), whereas the vast majority is located elsewhere in the chromatin
We have no evidence that topo I occurs close enough to ORC on chromatin to be coprecipitated with ORC on micrococcal nuclease (MN) digestion products, or to be cross-linked together with ORC on chromatin fragments of ca. 500 bp
Summary
The process of DNA replication requires the separation of complementary DNA strands In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. According to earlier biochemical work, high local concentrations of the eukaryotic type IB topoisomerase (topo I) and a type II topoisomerase (topo II) are required to release the torsional stress that accompanies the initiation and propagation of replication forks on closed circular SV40 viral DNA in vitro [1,2,3]. BMC Molecular Biology 2009, 10:36 http://www.biomedcentral.com/1471-2199/10/36 replicated DNA circles [4,5,6,7] Both eukaryotic topo I and topo II have the properties to release the positive supercoils that form ahead of the advancing replication forks, and the negative supercoils that accumulate in the replicated DNA sections [8,9]
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