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Nucleic Acids Research 2006 34(13):e91; doi:10.1093/nar/gkl512
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Published online 26 July 2006

Nucleic Acids Research, 2006, Vol. 34, No. 13 e91
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

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De novo assembly of genuine replication forks on an immobilized circular plasmid in Xenopus egg extracts

Akiko Zembutsu and Shou Waga1,*

Department of Biology, Graduate School of Science, Osaka University Toyonaka, Osaka 560-0043, Japan 1 Laboratories for Biomolecular Network, Graduate School of Frontier Biosciences, Osaka University Suita, Osaka 565-0871, Japan

*To whom correspondence should be addressed. Tel/Fax: +81 6 6879 4660; Email: swaga{at}fbs.osaka-u.ac.jp

Received April 24, 2006. Revised July 6, 2006. Accepted July 6, 2006.

We describe an improved model of DNA replication in Xenopus egg extracts, in which a circular plasmid immobilized on paramagnetic beads is used as a template. DNA synthesis occurred on either circular or linear plasmids coupled to the beads, but only DNA synthesis on the circular plasmid was inhibited by geminin and a CDK inhibitor, p21. DNA synthesis on the circular plasmid occurred after a time lag, during which nuclear formation was probably occurring. Although pre-replicative complexes (pre-RCs) were formed soon after mixing plasmids with egg extracts, binding of CDC45, RPA, Pol {alpha}, {delta} and {varepsilon}, and PCNA to the circular plasmid was delayed, but still correlated with DNA synthesis. Moreover, p21 inhibited binding of these replication fork proteins to the circular plasmid. Therefore, the circular plasmid, but not the linear plasmid, assembles bona fide replication forks in egg extracts. We conclude that this improved replication system will be useful for studying the mechanism of formation of replication forks in eukaryotic DNA replication.


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