Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos

Chromosome replication initiates without sequence specificity at average in tervals of similar to 10 kb during the rapid cell cycles of early Xenopus e mbryos. If the distribution of origins were random, some inter-origin inter vals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have exam ined the replication intermediates of plasmids of various sizes (5.3-42.2 k bp) in Xenopus egg extracts by two-dimensional gel electrophoresis and elec tron microscopy. We confirm that replication initiates without sequence spe cificity on all plasmids. We demonstrate for the first time that multiple i nitiation events occur on large plasmids, but not on small (<10 kb) plasmid s, at average intervals of similar to 10 kb. Origin interference may preven t multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on l ater than on earlier replication intermediates, showing that initiation fre quency increases throughout S phase, speeding up replication of late interm ediates. We suggest that potential origins are abundant and randomly distri buted, but that the increase of initiation frequency during S phase, and po ssibly origin interference, regulate origin activation to ensure rapid comp letion of replication. (C) 2000 Academic Press.