Abstract
An in vitro system for mammalian origin-rich sequences(ors) has been developed. The system uses HeLa cell extracts and is dependent upon the presence of an ors template. In vitro replication has been shown to initiate in the ors, is semi-conservative, bidirectional, aphidicolin-sensitive, and independent of SV40 T-antigen. This in vitro system affords an opportunity to study the essential factors necessary for initiation of replication from mammalian ors. One structural feature which we have studied as potential contributor to the regulation of DNA replication is an extruded inverted repeat yielding a cruciform structure. Monoclonal antibodies specific for cruciform DNA, reacting with the “elbow” at the base of the stem-loop of the cruciform, have been prepared and tested in permeablized nuclei and in this in vitro system of mammalian DNA replication. Addition of anti-cruciform antibody to the in vitro reaction mixture results in 1 to 2 orders of magnitude enhancement of overall in vitro DNA synthesis.
In the following discussion we will describe the essential methodology for establishment of an in vitro replication system for mammalian ors and summarize some of our experience with the the characterization of the system.
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References
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Price, G.B., Pearson, C.E., Zannis-Hadjopoulos, M. (1992). An in Vitro Replication System for Autonomously Replicating Mammalian Origin-Enriched Sequences. In: Poirier, G.G., Moreau, P. (eds) ADP-Ribosylation Reactions. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-8718-1_21
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DOI: https://doi.org/10.1007/978-1-4419-8718-1_21
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