Replication origins in Xenopus egg extract are 5-15 kilobases apart and are activated in clusters that fire at different times

When Xenopus eggs and egg extracts replicate DNA, replication origins are p ositioned randomly with respect to DNA sequence. However, a completely rand om distribution of origins would generate some unacceptably large interorig in distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [H-3]thymidine or bromodeoxyuridine and the geometry of l abeled sites on spread DNA was examined. Most origins were spaced 5-15 kb a part. This regular distribution provides an explanation for how complete ch romosome replication can be ensured although origins are positioned randoml y with respect to DNA sequence. Origins weregrouped into small clusters (ty pically containing 5-10 replicons) that fired at approximately the same tim e, with different clusters being activated at different times in S phase. T his 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 or igin recognition complexes (ORCs) on the chromatin was restricted, the aver age interorigin distance increased, and the number of origins in each clust er decreased. This suggests that the binding of ORCs to chromatin determine s the regular spacing of origins in this system.