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© The Rockefeller University Press, 0021-9525/2001//15 $5.00
The Journal of Cell Biology, Volume 152, Number 1, , 2001 15-26

Replication Origins in XenopusEgg Extract Are 5–15 Kilobases Apart and Are Activated in Clusters That Fire at Different Times

^a Cancer Research Campaign, Chromosome Replication Research Group, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, United Kingdom
^b School of Biosciences, Cardiff University, Cardiff CF1 3TL, United Kingdom
^c University of Manchester Institute of Science and Technology, Department of Biomolecular Sciences, Manchester M60 1QD, United Kingdom
^d Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
Department of Biochemistry, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK.(44) 1382-348072(44) 1382-345797

j.j.blow{at}dundee.ac.uk

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [³H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5–15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5–10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

Key Words: replication origin • Xenopus • S phase • ORC • origin clusters

© 2001 The Rockefeller University Press

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